Yeast-based vaccines as immunotherapy

ABSTRACT

Compositions and methods for treating and/or preventing a variety of diseases and conditions that are amenable to immunotherapy and, in one particular embodiment, compositions and methods for treating and/or preventing cancer in an animal are described. Specifically improvements related to the use of a yeast-based vaccine comprising a yeast vehicle and an antigen that is selected to elicit an antigen-specific cellular and humoral immune response in an animal, for use in prophylactic and/or therapeutic vaccination and the prevention and/or treatment of a variety of diseases and conditions are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/738,646, filed Dec. 16, 2003, now U.S. Pat. No. 7,465,454, whichclaims the benefit of priority under 35 U.S.C. §119(e) from U.S.Provisional Application Ser. No. 60/434,163, filed Dec. 16, 2002, andentitled “Yeast-Based Vaccines as Cancer Immunotherapy”. The entiredisclosure of each of U.S. Provisional Application Ser. No. 60/434,163and U.S. patent application Ser. No. 10/738,646 is incorporated hereinby reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-6-4_ST25”, has a sizein bytes of 35 KB, and was recorded on 5 Dec. 2008. The informationcontained in the text file is incorporated herein by reference in itsentirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to the use of yeast-basedvaccines comprising heterologous antigens for the elicitation of humoraland cellular immunity and in one aspect, for the prevention andtreatment of a variety of cancers in an animal.

BACKGROUND OF THE INVENTION

Neoplasia, or a process of rapid cellular proliferation resulting innew, abnormal growth, is a characteristic of many diseases which can beserious, and sometimes, life-threatening. Typically, neoplastic growthof cells and tissues is characterized by greater than normalproliferation of cells, wherein the cells continue to grow even afterthe instigating factor (e.g., tumor promoter, carcinogen, virus) is nolonger present. The cellular growth tends to show a lack of structuralorganization and/or coordination with the normal tissue and usuallycreates a mass of tissue (e.g., a tumor) which may be benign ormalignant. Malignant cellular growth, or malignant tumors, are a leadingcause of death worldwide, and the development of effective therapy forneoplastic disease is the subject of a large body of research. Althougha variety of innovative approaches to treat and prevent cancers havebeen proposed, many cancers continue to have a high rate of mortalityand may be difficult to treat or relatively unresponsive to conventionaltherapies.

For example, lung cancer is the second most common form of cancer in theUnited States. It accounts for 15% of all cancers and 28% of all cancerdeaths. In 2002 an estimated 177,000 new cases will be diagnosed and166,000 will die, a mortality rate higher than colorectal, prostate andbreast combined. 80% of primary lung tumors are non-small cell lungcarcinoma (NSCLC). Standard chemotherapy continues to be relativelyineffective with multiple drug therapy yielding minimal survivaladvantage with significant toxicity.

As another example, glioblastoma multiforme (glioma) is the most commonprimary malignant brain tumor in adults. Despite the use of surgery,radiotherapy and chemotherapy, cure rates and median patient survivalhave not improved. Other tumors also metastasize to the brain and inthis setting they respond less well to peripheral chemotherapy due toconstraints on drug delivery imposed by the blood/brain barrier.Clearly, more brain tumor-directed therapeutic approaches are needed.One such approach involves immunotherapy. It has been known for sometime that lymphocytes primed in the periphery can traverse the bloodbrain barrier and target brain tissue. Prime targets for brain tumorimmunotherapy are vaccines that elicit immune responses against new ormutated antigens expressed specifically in brain tumor cells. The goalthen is to provide a vaccine approach that would provide broad, vigorousand long-lasting immune protection against intracranial tumors.

Vaccines are widely used to prevent disease and to treat establisheddiseases (immunotherapeutic vaccines). Protein antigens (e.g. subunitvaccines, the development of which was made possible by recombinant DNAtechnology), when administered without adjuvants, induce weak humoral(antibody) immunity and have therefore been disappointing to date asthey generate only limited immunogenicity. An additional disadvantage ofsubunit vaccines, as well as of killed virus and recombinant live virusvaccines, is that while they appear to stimulate a strong humoral immuneresponse when administered with adjuvants, they fail to elicitprotective cellular immunity. Adjuvants are used experimentally tostimulate potent immune responses in mice, and are desirable for use inhuman vaccines, but few are approved for human use. Indeed, the onlyadjuvants approved for use in the United States are the aluminum salts,aluminum hydroxide and aluminum phosphate, neither of which stimulatescell-mediated immunity. Aluminum salt formulations cannot be frozen orlyophilized, and such adjuvants are not effective with all antigens.Moreover, most adjuvants do not lead to induction of cytotoxic Tlymphocytes (CTL). CTL are needed to kill cells that are synthesizingaberrant proteins including viral proteins and mutated “self” proteins.Vaccines that stimulate CTL are being intensely studied for use againsta variety of diseases, including all cancers (e.g., melanoma, prostate,ovarian, etc.). Thus adjuvants are needed that stimulate CTL andcell-mediated immunity in general.

Yeast have been used in the production of subunit protein vaccines;however, in this case, yeast are used to produce the protein, but theyeast cells or subcellular fractions thereof are not actually deliveredto the patient. Yeast have also been fed to animals prior toimmunization to try to prime the immune response in a non-specificmanner (i.e., to stimulate phagocytosis as well as the production ofcomplement and interferon). The results have been ambiguous, and suchprotocols have not generated protective cellular immunity; see, forexample, Fattal-German et al., 1992, Dev. Biol. Stand. 77, 115-120;Bizzini et al., 1990, FEMS Microbiol. Immunol. 2, 155-167.

U.S. Pat. No. 5,830,463, issued Nov. 3, 1998, to Duke et al. describedthe use of nonpathogenic yeast carrying at least one compound capable ofmodulating an immune response, and demonstrated that such complexes areefficacious at stimulating cell-mediated, as well as humoral, immunity.In particular, U.S. Pat. No. 5,830,463 demonstrated that yeast which aregenetically engineered to express a heterologous antigen can elicit botha cell-mediated and a humoral immune response when administered to ananimal.

Despite the current advances in cancer therapy and vaccine technology,there remains an urgent need to develop safe and effective vaccines andadjuvants for diseases that are amenable to immunotherapy, includingdisease caused by neoplastic transformation (cancer), and particularly,for those cancers that are especially resistant to treatment usingconventional cancer therapy and generic vaccine strategies.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method to protectan animal against a cancer, comprising administering to an animal thathas or is at risk of developing a cancer, a vaccine to reduce or preventat least one symptom of the cancer in the animal. The vaccine comprises:(a) a yeast vehicle; and (b) a fusion protein expressed by the yeastvehicle, the fusion protein comprising: (i) at least one cancer antigen;and (ii) a peptide linked to the N-terminus of the cancer antigen, thepeptide consisting of at least two amino acid residues that areheterologous to the cancer antigen, wherein the peptide stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. Thefusion protein has the following additional requirements: (1) the aminoacid residue at position one of the fusion protein is a methionine; (2)the amino acid residue at position two of the fusion protein is not aglycine or a proline; (3) none of the amino acid residues at positions2-6 of the fusion protein is a methionine; and, (4) none of the aminoacid residues at positions 2-5 of the fusion protein is a lysine or anarginine. In one aspect, the peptide consists of at least 2-6 amino acidresidues that are heterologous to the cancer antigen. In another aspect,the peptide comprises an amino acid sequence of M-X₂-X₃-X₄-X₅-X₆,wherein X₂ is any amino acid except glycine, proline, lysine orarginine; wherein X₃ is any amino acid except methionine, lysine orarginine; wherein X₄ is any amino acid except methionine, lysine orarginine; wherein X₅ is any amino acid except methionine, lysine orarginine; and wherein X₆ is any amino acid except methionine. In oneaspect, X₆ is a proline. In another aspect, the peptide comprises anamino acid sequence of M-A-D-E-A-P (SEQ ID NO:1).

Another embodiment of the present invention relates to a method toprotect an animal against a cancer, comprising administering to ananimal that has or is at risk of developing a cancer, a vaccine toreduce or prevent at least one symptom of the cancer in the animal. Thevaccine comprises: (a) a yeast vehicle; and (b) a fusion proteinexpressed by the yeast vehicle, the fusion protein comprising: (i) atleast one cancer antigen; and (ii) a yeast protein linked to theN-terminus of the cancer antigen, wherein the yeast protein consists ofbetween about two and about 200 amino acids of an endogenous yeastprotein, wherein the yeast protein stabilizes the expression of thefusion protein in the yeast vehicle or prevents posttranslationalmodification of the expressed fusion protein. In one aspect, the yeastprotein comprises an antibody epitope for identification andpurification of the fusion protein.

In either of the above-described embodiments of the invention, thefollowing additional aspects are contemplated. In one aspect, the fusionprotein comprises at least two or more cancer antigens. In anotheraspect, the fusion protein comprises at least one or more immunogenicdomain of one or more cancer antigens. In another aspect, the cancerantigen is an antigen associated with a cancer selected from the groupconsisting of: melanomas, squamous cell carcinoma, breast cancers, headand neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bonesarcomas, testicular cancers, prostatic cancers, ovarian cancers,bladder cancers, skin cancers, brain cancers, angiosarcomas,hemangiosarcomas, mast cell tumors, primary hepatic cancers, lungcancers, pancreatic cancers, gastrointestinal cancers, renal cellcarcinomas, hematopoietic neoplasias and metastatic cancers thereof.

In yet another aspect, the cancer antigen is wild-type or mutant proteinencoded by a ras gene. For example, the cancer antigen can include awild-type or mutant protein encoded by a ras gene selected from thegroup consisting of: K-ras, N-ras and H-ras genes. In one aspect, theras gene encodes a Ras protein with single or multiple mutations. Inanother aspect, the cancer antigen comprises fragments of at least 5-9contiguous amino acid residues of a wild-type Ras protein containingamino acid positions 12, 13, 59 or 61 relative to the wild-type Rasprotein, wherein the amino acid residues at positions 12, 13, 59 or 61are mutated with respect to the wild-type Ras protein.

In yet another aspect, the cancer antigen consists of a fusion proteinconstruct comprising multiple domains, wherein each domain consists of apeptide from an oncoprotein, the peptide consisting of at least 4 aminoacid residues flanking either side of and including a mutated amino acidthat is found in the protein, wherein the mutation is associated withtumorigenicity. In this aspect, the fusion protein construct consists ofat least one peptide that is fused in frame with another mutated tumorantigen, wherein the peptide is selected from the group consisting of:(a) a peptide comprising at least from positions 8-16 of SEQ ID NO:3,wherein the amino acid residue at position 12 with respect to SEQ IDNO:3 is mutated as compared to SEQ ID NO:3; (b) a peptide comprising atleast from positions 9-17 of SEQ ID NO:3, wherein the amino acid residueat position 13 with respect to SEQ ID NO:3 is mutated as compared to SEQID NO:3; (c) a peptide comprising at least from positions 55-63 of SEQID NO:3, wherein the amino acid residue at position 59 with respect toSEQ ID NO:3 is mutated as compared to SEQ ID NO:3; and (d) a peptidecomprising at least from positions 57-65 of SEQ ID NO:3, wherein theamino acid residue at position 61 with respect to SEQ ID NO:3 is mutatedas compared to SEQ ID NO:3. In one aspect, the mutated tumor antigen isa Ras protein comprising at least one mutation relative to a wild-typeRas protein sequence.

In one embodiment of either of the above-identified methods, the vaccineis administered to the respiratory tract. In another embodiment, thevaccine is administered by a parenteral route of administration. In yetanother embodiment, the vaccine further comprises dendritic cells ormacrophages, wherein the yeast vehicle expressing the fusion protein isdelivered to dendritic cells or macrophages ex vivo and wherein thedendritic cell or macrophage containing the yeast vehicle expressing thecancer antigen is administered to the animal. In one aspect of thisembodiment, the dendritic cell or the yeast vehicle has beenadditionally loaded with free antigen. In one aspect, the vaccine isadministered as a therapeutic vaccine. In another aspect, the vaccine isadministered as a prophylactic vaccine. In one aspect, the animal has oris at risk of developing a cancer selected from the group consisting ofbrain cancer, lung cancer, breast cancer, melanoma, and renal cancer. Inanother aspect, the animal has cancer and wherein administration of thevaccine occurs after surgical resection of a tumor from the animal. Inyet another aspect, the animal has cancer and wherein administration ofthe vaccine occurs after surgical resection of a tumor from the animaland after administration of non-myeloablative allogeneic stem celltransplantation. In yet another aspect, the animal has cancer andwherein administration of the vaccine occurs after surgical resection ofa tumor from the animal, after administration of non-myeloablativeallogeneic stem cell transplantation, and after allogeneic donorlymphocyte infusion.

Another embodiment of the invention relates to a method to protect ananimal against a brain cancer or a lung cancer, comprising administeringto the respiratory tract of an animal that has or is at risk ofdeveloping a brain cancer or a lung cancer, a vaccine comprising a yeastvehicle and at least one cancer antigen, to reduce or prevent at leastone symptom of the brain cancer or lung cancer in the animal. In thisembodiment, the vaccine can include any of the above-described fusionproteins, as well as other antigens. In one aspect, the vaccinecomprises at least two or more cancer antigens. In another aspect, thecancer antigen is a fusion protein comprising at least one or morecancer antigens. In yet another aspect, the cancer antigen is a fusionprotein comprising at least one or more immunogenic domains of one ormore cancer antigens.

In one aspect of this embodiment, the vaccine is administered byintranasal administration. In another aspect, the vaccine isadministered by intratracheal administration. In yet another embodiment,the yeast vehicle and the cancer antigen are delivered to dendriticcells or macrophages ex vivo and wherein the dendritic cell ormacrophage containing the yeast vehicle and cancer antigen areadministered to the respiratory tract of the animal.

In one aspect, the method protects the animal against a brain cancer,including, but not limited to a primary brain cancer, such as aglioblastoma multiforme, or a metastatic cancer from a different organ.In another embodiment, the method protects the animal against a lungcancer, including, but not limited to a primary lung cancer (e.g.,non-small cell carcinomas, small cell carcinomas and adenocarcinomas) ora metastatic cancer from a different organ. In one aspect, the vaccineis administered as a therapeutic vaccine. In another aspect, the vaccineis administered as a prophylactic vaccine.

Yet another embodiment of the present invention relates to a method toelicit an antigen-specific humoral immune response and anantigen-specific cell-mediated immune response in an animal. The methodincludes administering to the animal a therapeutic compositioncomprising: (a) a yeast vehicle; and (b) a fusion protein expressed bythe yeast vehicle, the fusion protein comprising: (i) at least oneantigen; and (ii) a peptide linked to the N-terminus of the antigen, thepeptide consisting of at least two amino acid residues that areheterologous to the antigen, wherein the peptide stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. Thefusion protein has the following additional requirements: the amino acidresidue at position one of the fusion protein is a methionine; the aminoacid residue at position two of the fusion protein is not a glycine or aproline; none of the amino acid residues at positions 2-6 of the fusionprotein is a methionine; and, none of the amino acid residues atpositions 2-5 of the fusion protein is a lysine or an arginine. In oneaspect, the peptide consists of at least six amino acid residues thatare heterologous to the antigen. In another aspect, the peptidecomprises an amino acid sequence of M-X₂-X₃-X₄-X₅-X₆: wherein X₂ is anyamino acid except glycine, proline, lysine or arginine; wherein X₃ isany amino acid except methionine, lysine or arginine; wherein X₄ is anyamino acid except methionine, lysine or arginine; wherein X₅ is anyamino acid except methionine, lysine or arginine; and wherein X₆ is anyamino acid except methionine. In one aspect, X₆ is a proline. In oneaspect, the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQID NO:1). In one aspect, the antigen is selected from the groupconsisting of: a viral antigen, an overexpressed mammalian cell surfacemolecule, a bacterial antigen, a fungal antigen, a protozoan antigen, ahelminth antigen, an ectoparasite antigen, a cancer antigen, a mammaliancell molecule harboring one or more mutated amino acids, a proteinnormally expressed pre- or neo-natally by mammalian cells, a proteinwhose expression is induced by insertion of an epidemiologic agent (e.g.virus), a protein whose expression is induced by gene translocation, anda protein whose expression is induced by mutation of regulatorysequences.

Another embodiment relates to a vaccine as described for use in themethod above.

Yet another embodiment of the invention relates to a method to elicit anantigen-specific humoral immune response and an antigen-specificcell-mediated immune response in an animal. The method includesadministering to the animal a therapeutic composition comprising: (a) ayeast vehicle; and (b) a fusion protein expressed by the yeast vehicle,the fusion protein comprising: (i) at least one antigen; and (ii) ayeast protein linked to the N-terminus of the antigen, wherein the yeastprotein consists of between about two and about 200 amino acids of anendogenous yeast protein, wherein the yeast protein stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. In oneaspect, the yeast protein comprises an antibody epitope foridentification and purification of the fusion protein.

Another embodiment of the invention is a vaccine as described for use inthe method above.

Yet another embodiment of the present invention relates to a methodtreat a patient that has cancer, comprising: (a) treating a patient thathas cancer by nonmyeloablative stem cell transfer effective to establisha stable mixed bone marrow chimerism, wherein the stem cells areprovided by an allogeneic donor; (b) administering lymphocytes obtainedfrom the allogeneic donor to the patient; and (c) administering to thepatient, after step (b), a vaccine comprising a yeast vehicle and atleast one cancer antigen. In one aspect, the method also includesadministering to the allogeneic donor, prior to step (a), a vaccinecomprising a yeast vehicle and at least one cancer antigen. In anotherembodiment, the method includes removing a tumor from the patient priorto performing step (a).

In one aspect of this method, the vaccine comprises at least two or morecancer antigens. In another aspect, the cancer antigen is a fusionprotein comprising one or more cancer antigens. In yet another aspect,the cancer antigen is a fusion protein comprising one or moreimmunogenic domains of one or more cancer antigens. In another aspect,the cancer antigen consists of a fusion protein construct comprisingmultiple domains, wherein each domain consists of a peptide from anoncoprotein, the peptide consisting of at least 4 amino acid residuesflanking either side of and including a mutated amino acid that is foundin the protein, wherein the mutation is associated with tumorigenicity.In another aspect, the yeast vehicle expresses the cancer antigen, andwherein the cancer antigen is a fusion protein comprising: (a) at leastone cancer antigen; and (b) a peptide linked to the N-terminus of thecancer antigen, the peptide consisting of at least two amino acidresidues that are heterologous to the cancer antigen, wherein thepeptide stabilizes the expression of the fusion protein in the yeastvehicle or prevents posttranslational modification of the expressedfusion protein: wherein the amino acid residue at position one of thefusion protein is a methionine; wherein the amino acid residue atposition two of the fusion protein is not a glycine or a proline;wherein none of the amino acid residues at positions 2-6 of the fusionprotein is a methionine; and, wherein none of the amino acid residues atpositions 2-5 of the fusion protein is a lysine or an arginine. Inanother aspect, the yeast vehicle expresses the cancer antigen, andwherein the cancer antigen is a fusion protein comprising: (a) at leastone cancer antigen; and (b) a yeast protein linked to the N-terminus ofthe cancer antigen, wherein the yeast protein consists of between abouttwo and about 200 amino acids of an endogenous yeast protein, whereinthe yeast protein stabilizes the expression of the fusion protein in theyeast vehicle or prevents posttranslational modification of theexpressed fusion protein.

In one aspect of this embodiment, the vaccine is administered byintranasal administration. In another aspect, the vaccine isadministered by parenteral administration. In another aspect, the yeastvehicle and the cancer antigen are delivered to dendritic cells ormacrophages ex vivo and wherein the dendritic cell or macrophagecontaining the yeast vehicle and cancer antigen are administered to therespiratory tract of the animal.

In any of the above-described methods and compositions of the presentinvention, the following aspects related to the yeast vehicle areincluded in the invention. In one embodiment, yeast vehicle is selectedfrom the group consisting of a whole yeast, a yeast spheroplast, a yeastcytoplast, a yeast ghost, and a subcellular yeast membrane extract orfraction thereof. In one aspect, a yeast cell or yeast spheroplast usedto prepare the yeast vehicle was transformed with a recombinant nucleicacid molecule encoding the antigen such that the antigen isrecombinantly expressed by the yeast cell or yeast spheroplast. In thisaspect, the yeast cell or yeast spheroplast that recombinantly expressesthe antigen is used to produce a yeast vehicle comprising a yeastcytoplast, a yeast ghost, or a subcellular yeast membrane extract orfraction thereof. In one aspect, the yeast vehicle is from anon-pathogenic yeast. In another aspect, the yeast vehicle is from ayeast selected from the group consisting of: Saccharomyces,Schizosaccharomyces, Kluveromyces, Hansenula, Candida and Pichia. In oneaspect, the Saccharomyces is S. cerevisiae.

In general, the yeast vehicle and antigen can be associated by anytechnique described herein. In one aspect, the yeast vehicle was loadedintracellularly with the cancer antigen. In another aspect, the cancerantigen was covalently or non-covalently attached to the yeast vehicle.In yet another aspect, the yeast vehicle and the cancer antigen wereassociated by mixing. In another aspect, the antigen is expressedrecombinantly by the yeast vehicle or by the yeast cell or yeastspheroplast from which the yeast vehicle was derived.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIG. 1 is a bar graph showing that the yeast-based Ras61-VAX vaccinecontrols preexisting, urethane-induced lung tumors in vivo.

FIG. 2 is a bar graph showing that yeast-based RasV-VAX vaccine providesspecific protection against lung tumor growth when administered bysubcutaneous and intranasal routes.

FIG. 3 is bar graph showing that a Gag-expressing yeast based vaccineprotects against intracranial tumors when the vaccine is administered byintranasal, but not subcutaneous, administration.

FIG. 4 is a survival graph showing that the yeast-based vaccineexpressing EGFR (EGFR-tm VAX) protects against challenge withintracranial tumors expressing EGFR when administered subcutaneously andintranasally.

FIG. 5 is a bar graph showing that vaccination with a yeast-basedvaccine expressing a breast tumor antigen in conjunction withnon-myeloablative allogeneic stem cell transplantation protects againsttumor challenge.

FIG. 6 is a bar graph showing that vaccination with a yeast-basedvaccine expressing a melanoma antigen protects against tumor challengewith melanoma tumors expressing the antigen.

FIG. 7 is a schematic drawing showing the construction of various mutantRas fusion proteins for use in a yeast-based vaccine of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to compositions and methods fortreating and/or preventing a variety of diseases and conditions that areamenable to immunotherapy and, in one particular embodiment, tocompositions and methods for treating and/or preventing cancer in ananimal. The invention includes the use of a yeast-based vaccinecomprising a yeast vehicle and an antigen that is selected to elicit anantigen-specific cellular and humoral immune response in an animal, foruse in prophylactic and/or therapeutic vaccination and the preventionand/or treatment of a variety of diseases and conditions. In particular,the inventors describe herein the use of yeast-based vaccines to reducetumors in a variety of different forms of cancer in vivo, including lungcancer, brain cancer, breast cancer, and renal cancer. Also describedherein are improvements to a yeast-based vaccine that are applicable notonly to cancer therapies, but to the treatment of a variety ofimmunotherapeutic methods and compositions.

The inventors have previously described a vaccine technology thatelicits potent cell-mediated immunity, including cytotoxic T cell (CTL)responses. The vaccine technology involves using yeast and derivativesthereof as a vaccine vector, wherein the yeast are engineered to expressor are otherwise loaded with relevant antigen(s) to elicit an immuneresponse against the antigen(s). This technology is generally describedin U.S. Pat. No. 5,830,463 and is incorporated herein by reference inits entirety. The present invention takes the existing yeast vaccinetechnology described in U.S. Pat. No. 5,830,463 and provides specificimprovements in a method to reduce cancer using yeast vehicles andselected cancer antigens, as well as new yeast vaccines comprising novelproteins that have enhanced stability, and methods of using the newyeast vaccines to treat any disease or condition for which elicitationof an immune response may have a therapeutic benefit. A generaldescription of yeast vaccines that can be used in various embodiments ofthe invention is also described in copending U.S. application Ser. No.09/991,363, which is incorporated by reference in its entirety.

In particular, the present inventors have discovered that, whilemultiple routes of immunization may be equivalently effective fordestroying tumors in the periphery, the yeast-based vaccine used in thepresent invention is able to prime effector cells that may be unique tothe lung. Therefore, although other routes of administration are stilleffective, administration of the yeast vaccines through the respiratorytract (e.g., intranasal, inhaled, intratracheal) provides a surprisinglyrobust immune response and anti-tumor effect that is not achieved usingother routes of administration investigated thus far. In particular, thepresent inventors have discovered that administration of the yeastvaccine to the respiratory tract is significantly better at reducingtumors in lung cancer than when the vaccine is administered to theperiphery. Perhaps even more surprising was the result that in braintumors, while administration of the yeast vaccine to the respiratorytract induced a potent anti-tumor response in all experimental modelsexamined thus far, peripheral administration of the vaccine(subcutaneous) was less effective at inducing an anti-tumor response inthe brain, and in at least one experimental model for brain cancer,peripheral administration failed to provide a significant anti-tumoreffect in the brain. Therefore, yeast-based vaccines of the presentinvention can prime unique immune effector cell precursors in the lungs,and such immune cells may be particularly effective for crossing theblood-brain barrier to influence the course of intracranial tumorgrowth. Without being bound by theory, the present inventors believethat the route of immunization may be an important component in thedesign of an effective vaccine for at least brain tumors and lungtumors. Because the yeast-based vaccine of the invention is extremelyfacile for multiple routes of immunization, the vaccine holds thepromise to uniquely provoke highly specialized immune responses withheretofore underappreciated potential for the treatment of some cancers.

The present inventors have also discovered that the use of the yeastvaccines of the present invention in a novel modification of a mixedallogeneic bone marrow chimera protocol previously described by Luzniket al. (Blood 101 (4): 1645-1652, 2003; incorporated herein by referencein its entirety) results in excellent induction of therapeutic immunityand anti-tumor responses in vivo. Significantly, this result can beachieved without the need to use whole tumor preparations from therecipient and without the need to enhance the vaccine with biologicalresponse modifiers, such as granulocyte-macrophage colony-stimulatingfactor (GM-CSF), and without the need for the use of conventionaladjuvants. In addition, the use of the yeast vehicle of the presentinvention provides extreme flexibility in the choice of the antigen andantigen combinations, and provides significant enhancements of cellularimmunity against the antigen. Moreover, the present invention providesadditional enhancement of the protocol by providing for the immunizationof the donor with the yeast vaccine of the invention in a controlled,selective manner.

In addition, the present inventors have developed improvements to theyeast-based vaccine technology using novel fusion proteins thatstabilize the expression of the heterologous protein in the yeastvehicle and/or prevent posttranslational modification of the expressedheterologous protein. Specifically, the inventors describe herein anovel construct for expression of heterologous antigens in yeast,wherein the desired antigenic protein(s) or peptide(s) are fused attheir amino-terminal end to: (a) a synthetic peptide; or (b) at least aportion of an endogenous yeast protein, wherein either fusion partnerprovides significantly enhanced stability of expression of the proteinin the yeast and/or a prevents post-translational modification of theproteins by the yeast cells. Also, the fusion peptides provide anepitope that can be designed to be recognized by a selection agent, suchas an antibody, and do not appear to negatively impact the immuneresponse against the vaccinating antigen in the construct. Such agentsare useful for the identification, selection and purification ofproteins useful in the invention.

In addition, the present invention contemplates the use of peptides thatare fused to the C-terminus of the antigen construct, particularly foruse in the selection and identification of the protein. Such peptidesinclude, but are not limited to, any synthetic or natural peptide, suchas a peptide tag (e.g., 6×His) or any other short epitope tag. Peptidesattached to the C-terminus of an antigen according to the invention canbe used with or without the addition of the N-terminal peptidesdiscussed above.

Finally, the present inventors describe herein novel fusion proteinantigens for use in a yeast-based vaccine that provide multipleimmunogenic domains from one or more antigens within the same construct.Such fusion proteins are particularly useful when it is desirable toencompass several different mutations and/or combinations of mutationsthat may occur at one or a few positions in the antigen in nature, in asingle vaccine construct. For example, it is known that there areseveral different mutations in the oncogenes of the ras gene family thatcan be associated with a tumor cell phenotype in nature. Mutations atthe codon encoding amino acid 12 in the Ras protein are found in 78% ofpancreatic cancers, 34% of colorectal cancers, 27% of non-small celllung carcinomas, and 24% of ovarian cancers. Different mutations atpositions 13, 59 and 61 are also found in a variety of cancers. Usingthe yeast-based vaccine approach, the present inventors describe hereinthe production of fusion proteins, including, but not limited to, fusionproteins based on ras mutations, that can capture several mutations atthe same position and/or different combinations of mutations at morethan one position, all within the same antigen vaccine.

As a general description of the methods and compositions used in thepresent invention, the vaccine and methods described herein integrateefficient antigen delivery with extremely effective T cell activation ina powerful vaccine formulation that does not require accessory adjuvantcomponents or biological mediators. The vaccine approach describedherein has many other attributes that make it an ideal vaccinecandidate, including, but not limited to, ease of construction, lowexpense of mass production, biological stability, and safety. No grosslyadverse effects of immunization with whole yeast were apparent at thetime of the initial vaccination or upon repeated administration ineither mice, rats, rabbits, pig-tailed macaques (Macaca nemestrina),rhesus macaques, or immunodeficient CB. 17^(scid) mice (unpublishedobservations). Moreover, as described in application Ser. No.09/991,363, supra, the ability of yeast-antigen complexes to maturedendritic cells into potent antigen presenting cells (APCs) whileefficiently delivering antigens into both MHC class-I and class-IIprocessing pathways indicates that yeast-based vaccine vectors willprovide a powerful strategy for the induction of cell-mediated immunitydirected against a variety of infectious diseases and cancer targets.Indeed, the data described herein and the advances for the yeast-basedvaccine technology continue to prove this general principle whileproviding significant improvements to the technology that have not beenpreviously appreciated.

According to the present invention, a yeast vehicle is any yeast cell(e.g., a whole or intact cell) or a derivative thereof (see below) thatcan be used in conjunction with an antigen in a vaccine or therapeuticcomposition of the invention, or as an adjuvant. The yeast vehicle cantherefore include, but is not limited to, a live intact yeastmicroorganism (i.e., a yeast cell having all its components including acell wall), a killed (dead) intact yeast microorganism, or derivativesthereof including: a yeast spheroplast (i.e., a yeast cell lacking acell wall), a yeast cytoplast (i.e., a yeast cell lacking a cell walland nucleus), a yeast ghost (i.e., a yeast cell lacking a cell wall,nucleus and cytoplasm), or a subcellular yeast membrane extract orfraction thereof (also referred to previously as a subcellular yeastparticle).

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., 1991, Meth. Enzymol. 194, 662-674, incorporated herein byreference in its entirety. Yeast cytoplasts are typically produced byenucleation of yeast cells. Such a method is described, for example, inCoon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein byreference in its entirety. Yeast ghosts are typically produced byresealing a permeabilized or lysed cell and can, but need not, containat least some of the organelles of that cell. Such a method isdescribed, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258,3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196,each of which is incorporated herein by reference in its entirety. Asubcellular yeast membrane extract or fraction thereof refers to a yeastmembrane that lacks a natural nucleus or cytoplasm. The particle can beof any size, including sizes ranging from the size of a natural yeastmembrane to microparticles produced by sonication or other membranedisruption methods known to those skilled in the art, followed byresealing. A method for producing subcellular yeast membrane extracts isdescribed, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194,662-674. One may also use fractions of yeast membrane extracts thatcontain yeast membrane portions and, when the antigen was expressedrecombinantly by the yeast prior to preparation of the yeast membraneextract, the antigen of interest.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention. Yeast are unicellular microorganisms that belong to one ofthree classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Whilepathogenic yeast strains, or nonpathogenic mutants thereof can be usedin accordance with the present invention, nonpathogenic yeast strainsare preferred. Preferred genera of yeast strains include Saccharomyces,Candida (which can be pathogenic), Cryptococcus, Hansenula,Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia,with Saccharomyces, Candida, Hansenula, Pichia and Schizosaccharomycesbeing more preferred, and with Saccharomyces being particularlypreferred. Preferred species of yeast strains include Saccharomycescerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candidakefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcusneoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis,Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, andYarrowia lipolytica. It is to be appreciated that a number of thesespecies include a variety of subspecies, types, subtypes, etc. that aremeant to be included within the aforementioned species. More preferredyeast species include S. cerevisiae, C. albicans, H. polymorpha, P.pastoris and S. pombe. S. cerevisiae is particularly preferred due to itbeing relatively easy to manipulate and being “Generally Recognized AsSafe” or “GRAS” for use as food additives (GRAS, FDA proposed Rule62FR18938, Apr. 17, 1997). One embodiment of the present invention is ayeast strain that is capable of replicating plasmids to a particularlyhigh copy number, such as a S. cerevisiae cir° strain.

In one embodiment, a preferred yeast vehicle of the present invention iscapable of fusing with the cell type to which the yeast vehicle andantigen is being delivered, such as a dendritic cell or macrophage,thereby effecting particularly efficient delivery of the yeast vehicle,and in many embodiments, the antigen, to the cell type. As used herein,fusion of a yeast vehicle with a targeted cell type refers to theability of the yeast cell membrane, or particle thereof, to fuse withthe membrane of the targeted cell type (e.g., dendritic cell ormacrophage), leading to syncytia formation. As used herein, a syncytiumis a multinucleate mass of protoplasm produced by the merging of cells.A number of viral surface proteins (including those of immunodeficiencyviruses such as HIV, influenza virus, poliovirus and adenovirus) andother fusogens (such as those involved in fusions between eggs andsperm) have been shown to be able to effect fusion between two membranes(i.e., between viral and mammalian cell membranes or between mammaliancell membranes). For example, a yeast vehicle that produces an HIVgp120/gp41 heterologous antigen on its surface is capable of fusing witha CD4+ T-lymphocyte. It is noted, however, that incorporation of atargeting moiety into the yeast vehicle, while it may be desirable undersome circumstances, is not necessary. The present inventors havepreviously shown that yeast vehicles of the present invention arereadily taken up by dendritic cells (as well as other cells, such asmacrophages).

Yeast vehicles can be formulated into compositions of the presentinvention, including preparations to be administered to a patientdirectly or first loaded into a carrier such as a dendritic cell, usinga number of techniques known to those skilled in the art. For example,yeast vehicles can be dried by lyophilization or frozen by exposure toliquid nitrogen or dry ice. Formulations comprising yeast vehicles canalso be prepared by packing yeast in a cake or a tablet, such as is donefor yeast used in baking or brewing operations. In addition, prior toloading into a dendritic cell, or other type of administration with anantigen, yeast vehicles can also be mixed with a pharmaceuticallyacceptable excipient, such as an isotonic buffer that is tolerated bythe host cell. Examples of such excipients include water, saline,Ringer's solution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.Other useful formulations include suspensions containing viscosityenhancing agents, such as sodium carboxymethylcellulose, sorbitol,glycerol or dextran. Excipients can also contain minor amounts ofadditives, such as substances that enhance isotonicity and chemicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includethimerosal, m- or o-cresol, formalin and benzyl alcohol. Standardformulations can either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Thus, in a non-liquid formulation, the excipient can comprise, forexample, dextrose, human serum albumin, and/or preservatives to whichsterile water or saline can be added prior to administration.

One component of a therapeutic composition or vaccine of the presentinvention includes at least one antigen for vaccinating an animal. Thecomposition or vaccine can include, one, two, a few, several or aplurality of antigens, including one or more immunogenic domains of oneor more antigens, as desired. According to the present invention, thegeneral use herein of the term “antigen” refers: to any portion of aprotein (peptide, partial protein, full-length protein), wherein theprotein is naturally occurring or synthetically derived, to a cellularcomposition (whole cell, cell lysate or disrupted cells), to an organism(whole organism, lysate or disrupted cells) or to a carbohydrate orother molecule, or a portion thereof, wherein the antigen elicits anantigen-specific immune response (humoral and/or cellular immuneresponse), or alternatively acts as a toleragen, against the same orsimilar antigens that are encountered within the cells and tissues ofthe animal to which the antigen is administered.

In one embodiment of the present invention, when it is desirable tostimulate an immune response, the term “antigen” can be usedinterchangeably with the term “immunogen”, and is used herein todescribe a protein, peptide, cellular composition, organism or othermolecule which elicits a humoral and/or cellular immune response (i.e.,is antigenic), such that administration of the immunogen to an animal(e.g., via a vaccine of the present invention) mounts anantigen-specific immune response against the same or similar antigensthat are encountered within the tissues of the animal. Therefore, tovaccinate an animal against a particular antigen means, in oneembodiment, that an immune response is elicited against the antigen as aresult of administration of the antigen. Vaccination preferably resultsin a protective or therapeutic effect, wherein subsequent exposure tothe antigen (or a source of the antigen) elicits an immune responseagainst the antigen (or source) that reduces or prevents a disease orcondition in the animal. The concept of vaccination is well known in theart. The immune response that is elicited by administration of atherapeutic composition of the present invention can be any detectablechange in any facet of the immune response (e.g., cellular response,humoral response, cytokine production), as compared to in the absence ofthe administration of the vaccine.

In another embodiment, when it is desirable to suppress an immuneresponse against a given antigen, an antigen can include a toleragen.According to the present invention, a toleragen is used to describe aprotein, peptide, cellular composition, organism or other molecule thatis provided in a form, amount, or route of administration such thatthere is a reduced or changed immune response to the antigen, andpreferably substantial non-responsiveness, anergy, other inactivation,or deletion of immune system cells in response to contact with thetoleragen or a cell expressing or presenting such toleragen.

A “vaccinating antigen” can be an immunogen or a toleragen, but is anantigen used in a vaccine, where a biological response (elicitation ofan immune response, tolerance) is to be elicited against the vaccinatingantigen.

An immunogenic domain of a given antigen can be any portion of theantigen (i.e., a peptide fragment or subunit) that contains at least oneepitope that acts as an immunogen when administered to an animal. Forexample, a single protein can contain multiple different immunogenicdomains.

An epitope is defined herein as a single immunogenic site within a givenantigen that is sufficient to elicit an immune response, or a singletoleragenic site within a given antigen that is sufficient to suppress,delete or render inactive an immune response. Those of skill in the artwill recognize that T cell epitopes are different in size andcomposition from B cell epitopes, and that epitopes presented throughthe Class I MHC pathway differ from epitopes presented through the ClassII MHC pathway. An antigen can be as small as a single epitope, orlarger, and can include multiple epitopes. As such, the size of anantigen can be as small as about 5-12 amino acids (e.g., a peptide) andas large as: a full length protein, including a multimer and fusionproteins, chimeric proteins, whole cells, whole microorganisms, orportions thereof (e.g., lysates of whole cells or extracts ofmicroorganisms). In addition, antigens include carbohydrates, such asthose expressed on cancer cells, which can be loaded into a yeastvehicle or into a composition of the invention. It will be appreciatedthat in some embodiments (i.e., when the antigen is expressed by theyeast vehicle from a recombinant nucleic acid molecule), the antigen isa protein, fusion protein, chimeric protein, or fragment thereof, ratherthan an entire cell or microorganism. In preferred embodiments, theantigen is selected from the group of a tumor antigen or an antigen ofan infectious disease pathogen (i.e., a pathogen antigen). In oneembodiment, the antigen is selected from the group of: a viral antigen,an overexpressed mammalian cell surface molecule, a bacterial antigen, afungal antigen, a protozoan antigen, a helminth antigen, an ectoparasiteantigen, a cancer antigen, a mammalian cell molecule harboring one ormore mutated amino acids, a protein normally expressed pre- orneo-natally by mammalian cells, a protein whose expression is induced byinsertion of an epidemiologic agent (e.g. virus), a protein whoseexpression is induced by gene translocation, and a protein whoseexpression is induced by mutation of regulatory sequences.

According to the present invention, an antigen suitable for use in thepresent composition or vaccine can include two or more immunogenicdomains or epitopes from the same antigen, two or more antigensimmunogenic domains, or epitopes from the same cell, tissue or organism,or two or more different antigens, immunogenic domains, or epitopes fromdifferent cells, tissues or organisms. Preferably, the antigen isheterologous to the yeast strain (i.e., is not protein that is naturallyproduced by the yeast strain in the absence of genetic or biologicalmanipulation).

One embodiment of the invention relates to several improved proteins foruse as antigens in the vaccines of the invention. Specifically, thepresent invention provides new fusion protein constructs that stabilizethe expression of the heterologous protein in the yeast vehicle and/orprevent posttranslational modification of the expressed heterologousprotein. These fusion proteins are most typically expressed asrecombinant proteins by the yeast vehicle (e.g., by an intact yeast oryeast spheroplast, which can optionally be further processed to a yeastcytoplast, yeast ghost, or yeast membrane extract or fraction thereof),although it is an embodiment of the invention that one or much suchfusion proteins could be loaded into a yeast vehicle or otherwisecomplexed or mixed with a yeast vehicle as described above to form avaccine of the present invention.

One such fusion construct useful in the present invention is a fusionprotein that includes: (a) at least one antigen (including immunogenicdomains and epitopes of a full-length antigen, as well as various fusionproteins and multiple antigen constructs as described elsewhere herein);and (b) a synthetic peptide. The synthetic peptide is preferably linkedto the N-terminus of the cancer antigen. This peptide consists of atleast two amino acid residues that are heterologous to the cancerantigen, wherein the peptide stabilizes the expression of the fusionprotein in the yeast vehicle or prevents posttranslational modificationof the expressed fusion protein. The synthetic peptide and N-terminalportion of the antigen together form a fusion protein that has thefollowing requirements: (1) the amino acid residue at position one ofthe fusion protein is a methionine (i.e., the first amino acid in thesynthetic peptide is a methionine); (2) the amino acid residue atposition two of the fusion protein is not a glycine or a proline (i.e.,the second amino acid in the synthetic peptide is not a glycine or aproline); (3) none of the amino acid residues at positions 2-6 of thefusion protein is a methionine (i.e., the amino acids at positions 2-6,whether part of the synthetic peptide or the protein, if the syntheticpeptide is shorter than 6 amino acids, do not include a methionine); and(4) none of the amino acids at positions 2-5 of the fusion protein is alysine or an arginine (i.e., the amino acids at positions 2-5, whetherpart of the synthetic peptide or the protein, if the synthetic peptideis shorter than 5 amino acids, do not include a lysine or an arginine).The synthetic peptide can be as short as two amino acids, but is morepreferably at least 2-6 amino acids (including 3, 4, 5 amino acids), andcan be longer than 6 amino acids, in whole integers, up to about 200amino acids.

In one embodiment, the peptide comprises an amino acid sequence ofM-X₂-X₃-X₄-X₅-X₆, wherein M is methionine; wherein X₂ is any amino acidexcept glycine, proline, lysine or arginine; wherein X₃ is any aminoacid except methionine, lysine or arginine; wherein X₄ is any amino acidexcept methionine, lysine or arginine; wherein X₅ is any amino acidexcept methionine, lysine or arginine; and wherein X₆ is any amino acidexcept methionine. In one embodiment, the X₆ residue is a proline. Anexemplary synthetic sequence that enhances the stability of expressionof an antigen in a yeast cell and/or prevents post-translationalmodification of the protein in the yeast includes the sequenceM-A-D-E-A-P (SEQ ID NO:1). In addition to the enhanced stability of theexpression product, the present inventors believe that this fusionpartner does not appear to negatively impact the immune response againstthe vaccinating antigen in the construct. In addition, the syntheticfusion peptides can be designed to provide an epitope that can berecognized by a selection agent, such as an antibody.

According to the present invention, “heterologous amino acids” are asequence of amino acids that are not naturally found (i.e., not found innature, in vivo) flanking the specified amino acid sequence, or that arenot related to the function of the specified amino acid sequence, orthat would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the specified amino acidsequence as it occurs in the gene, if such nucleotides in the naturallyoccurring sequence were translated using standard codon usage for theorganism from which the given amino acid sequence is derived. Therefore,at least two amino acid residues that are heterologous to the cancerantigen are any two amino acid residues that are not naturally foundflanking the cancer antigen.

Another embodiment of the present invention relates to a fusion proteinthat includes: (a) at least one antigen (including immunogenic domainsand epitopes of a full-length antigen, as well as various fusionproteins and multiple antigen constructs as described elsewhere herein)that is fused to (b) at least a portion of an endogenous yeast protein.The endogenous yeast protein is preferably fused to the N-terminal endof the cancer antigen(s) and provides significantly enhanced stabilityof expression of the protein in the yeast and/or a preventspost-translational modification of the proteins by the yeast cells. Inaddition, the endogenous yeast antigen, as with the synthetic peptide,this fusion partner does not appear to negatively impact the immuneresponse against the vaccinating antigen in the construct. Antibodiesmay already be available that selectively bind to the endogenous antigenor can be readily generated. Finally, if it is desired to direct aprotein to a particular cellular location (e.g., into the secretorypathway, into mitochondria, into the nucleus), then the construct canuse the endogenous signals for the yeast protein to be sure that thecellular machinery is optimized for that delivery system.

The endogenous yeast protein consists of between about two and about 200amino acids (or 22 kDa maximum) of an endogenous yeast protein, whereinthe yeast protein stabilizes the expression of the fusion protein in theyeast vehicle or prevents posttranslational modification of theexpressed fusion protein. Any suitable endogenous yeast protein can beused in this embodiment, and particularly preferred proteins include,but are not limited to, SUC2 (yeast invertase; which is a good candidatefor being able to express a protein both cytosolically and directing itinto the secretory pathway from the same promoter, but is dependent onthe carbon source in the medium); alpha factor signal leader sequence;SEC7; CPY; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinasePGK and triose phosphate isomerase TPI gene products for theirrepressible expression in glucose and cytosolic localization; Cwp2p forits localization and retention in the cell wall; the heat shock proteinsSSA1, SSA3, SSA4, SSC1 and KAR2, whose expression is induced and whoseproteins are more thermostable upon exposure of cells to heat treatment;the mitochondrial protein CYC1 for import into mitochondria; BUD genesfor localization at the yeast cell bud during the initial phase ofdaughter cell formation; ACT1 for anchoring onto actin bundles.

In one embodiment, the endogenous yeast protein/peptide or the syntheticpeptide comprises an antibody epitope for identification andpurification of the fusion protein. Preferably, an antibody is availableor produced that selectively binds to the fusion partner. According tothe present invention, the phrase “selectively binds to” refers to theability of an antibody, antigen binding fragment or binding partner ofthe present invention to preferentially bind to specified proteins. Morespecifically, the phrase “selectively binds” refers to the specificbinding of one protein to another (e.g., an antibody, fragment thereof,or binding partner to an antigen), wherein the level of binding, asmeasured by any standard assay (e.g., an immunoassay), is statisticallysignificantly higher than the background control for the assay. Forexample, when performing an immunoassay, controls typically include areaction well/tube that contain antibody or antigen binding fragmentalone (i.e., in the absence of antigen), wherein an amount of reactivity(e.g., non-specific binding to the well) by the antibody or antigenbinding fragment thereof in the absence of the antigen is considered tobe background. Binding can be measured using a variety of methodsstandard in the art including enzyme immunoassays (e.g., ELISA),immunoblot assays, etc.).

Antibodies are characterized in that they comprise immunoglobulindomains and as such, they are members of the immunoglobulin superfamilyof proteins. Isolated antibodies of the present invention can includeserum containing such antibodies, or antibodies that have been purifiedto varying degrees. Whole antibodies of the present invention can bepolyclonal or monoclonal. Alternatively, functional equivalents of wholeantibodies, such as antigen binding fragments in which one or moreantibody domains are truncated or absent (e.g., Fv, Fab, Fab′, or F(ab)₂fragments), as well as genetically-engineered antibodies or antigenbinding fragments thereof, including single chain antibodies orantibodies that can bind to more than one epitope (e.g., bi-specificantibodies), or antibodies that can bind to one or more differentantigens (e.g., bi- or multi-specific antibodies), may also be employedin the invention.

Generally, in the production of an antibody, a suitable experimentalanimal, such as, for example, but not limited to, a rabbit, a sheep, ahamster, a guinea pig, a mouse, a rat, or a chicken, is exposed to anantigen against which an antibody is desired. Typically, an animal isimmunized with an effective amount of antigen that is injected into theanimal. An effective amount of antigen refers to an amount needed toinduce antibody production by the animal. The animal's immune system isthen allowed to respond over a pre-determined period of time. Theimmunization process can be repeated until the immune system is found tobe producing antibodies to the antigen. In order to obtain polyclonalantibodies specific for the antigen, serum is collected from the animalthat contains the desired antibodies (or in the case of a chicken,antibody can be collected from the eggs). Such serum is useful as areagent. Polyclonal antibodies can be further purified from the serum(or eggs) by, for example, treating the serum with ammonium sulfate.

Monoclonal antibodies may be produced according to the methodology ofKohler and Milstein (Nature 256:495-497, 1975). For example, Blymphocytes are recovered from the spleen (or any suitable tissue) of animmunized animal and then fused with myeloma cells to obtain apopulation of hybridoma cells capable of continual growth in suitableculture medium. Hybridomas producing the desired antibody are selectedby testing the ability of the antibody produced by the hybridoma to bindto the desired antigen.

The invention also extends to non-antibody polypeptides, sometimesreferred to as binding partners, that have been designed to bindspecifically to, and either activate or inhibit as appropriate, aprotein of the invention. Examples of the design of such polypeptides,which possess a prescribed ligand specificity are given in Beste et al.(Proc. Natl. Acad. Sci. 96:1898-1903, 1999), incorporated herein byreference in its entirety.

In yet another embodiment of the invention, the antigen portion of thevaccine is produced as a fusion protein comprising two or more antigens.In one aspect, the fusion protein can include two or more immunogenicdomains or two or more epitopes of one or more antigens. In aparticularly preferred embodiment, the fusion protein comprises two ormore immunogenic domains, and preferably, multiple domains, of anantigen, wherein the multiple domains together encompass severaldifferent mutations and/or combinations of mutations that may occur atone or a few positions in the antigen in nature. This provides aparticular advantage of being capable of providing a vaccine against avery specific antigen that is known to be variably mutated in a varietyof patients. Such a vaccine may provide antigen-specific immunization ina broad range of patients. For example, a multiple domain fusion proteinuseful in the present invention may have multiple domains, wherein eachdomain consists of a peptide from a particular protein, the peptideconsisting of at least 4 amino acid residues flanking either side of andincluding a mutated amino acid that is found in the protein, wherein themutation is associated with a particular disease (e.g., cancer).

Ras is one example of an oncogene in which several mutations are knownto occur at particular positions and be associated with the developmentof one or more types of cancer. Therefore, one can construct fusionproteins that consist of peptides containing a particular residue thatis known to be mutated in certain cancers, wherein each domain containsa different mutation at that site in order to cover several or all knownmutations at that site. For example, with regard to Ras, one may provideimmunogenic domains comprising at least 4 amino acids on either side ofand including position 12, wherein each domain has a differentsubstitution for the glycine that normally occurs in the non-mutated Rasprotein. In one example, the cancer antigen comprises fragments of atleast 5-9 contiguous amino acid residues of a wild-type Ras proteincontaining amino acid positions 12, 13, 59 or 61 relative to thewild-type Ras protein, wherein the amino acid residues at positions 12,13, 59 or 61 are mutated with respect to the wild-type Ras protein. Inone aspect, the fusion protein construct consists of at least onepeptide that is fused in frame with another mutated tumor antigen (e.g.,a Ras protein comprising at least one mutation relative to a wild-typeRas protein sequence), wherein the peptide is selected from the groupconsisting of: (a) a peptide comprising at least from positions 8-16 ofSEQ ID NO: 3, wherein the amino acid residue at position 12 with respectto SEQ ID NO:3 is mutated as compared to SEQ ID NO:3; (b) a peptidecomprising at least from positions 9-17 of SEQ ID NO:3, wherein theamino acid residue at position 13 with respect to SEQ ID NO:3 is mutatedas compared to SEQ ID NO:3; (c) a peptide comprising at least frompositions 55-63 of SEQ ID NO:3, wherein the amino acid residue atposition 59 with respect to SEQ ID NO:3 is mutated as compared to SEQ IDNO:3; and (d) a peptide comprising at least from positions 57-65 of SEQID NO:3, wherein the amino acid residue at position 61 with respect toSEQ ID NO:3 is mutated as compared to SEQ ID NO:3. It is noted thatthese positions also correspond to any of SEQ ID NOs: 5, 7, 9, 11 or 13,since human and mouse sequences are identical in this region of theprotein and since K-Ras, H-Ras and N-Ras are identical in this region.

Other antigens for which such strategies can be particularly useful inthe present invention will be apparent to those of skill in the art andinclude, but are not limited to: any oncogene, TP53 (also known as p53),p73, BRAF, APC, Rb-1, Rb-2, VHL, BRCA1, BRCA2, AR (androgen receptor),Smad4, MDR1, and/or Flt-3.

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” the specified amino acid sequence. As discussed above,according to the present invention, the heterologous amino acids are asequence of amino acids that are not naturally found (i.e., not found innature, in vivo) flanking the specified amino acid sequence, or that arenot related to the function of the specified amino acid sequence, orthat would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the specified amino acidsequence as it occurs in the gene, if such nucleotides in the naturallyoccurring sequence were translated using standard codon usage for theorganism from which the given amino acid sequence is derived. Similarly,the phrase “consisting essentially of”, when used with reference to anucleic acid sequence herein, refers to a nucleic acid sequence encodinga specified amino acid sequence that can be flanked by from at leastone, and up to as many as about 60, additional heterologous nucleotidesat each of the 5′ and/or the 3′ end of the nucleic acid sequenceencoding the specified amino acid sequence. The heterologous nucleotidesare not naturally found (i.e., not found in nature, in vivo) flankingthe nucleic acid sequence encoding the specified amino acid sequence asit occurs in the natural gene or do not encode a protein that impartsany additional function to the protein or changes the function of theprotein having the specified amino acid sequence.

Tumor antigens useful in the present invention can include a tumorantigen such as a protein, glycoprotein or surface carbohydrates from atumor cell, an epitope from a tumor antigen, an entire tumor cell,mixtures of tumor cells, and portions thereof (e.g., lysates). In oneembodiment, tumor antigens useful in the present invention can beisolated or derived from an autologous tumor sample. An autologous tumorsample is derived from the animal to whom the therapeutic composition isto be administered. Therefore, such antigens will be present in thecancer against which an immune response is to be elicited. In oneaspect, the tumor antigen provided in a vaccine is isolated or derivedfrom at least two, and preferably from a plurality of allogeneic tumorsamples of the same histological tumor type. According to the presentinvention, a plurality of allogeneic tumor samples are tumor samples ofthe same histological tumor type, isolated from two or more animals ofthe same species who differ genetically at least within the majorhistocompatibility complex (MHC), and typically at other genetic loci.Therefore, if administered together, the plurality of tumor antigens canbe representative of the substantially all of the tumor antigens presentin any of the individuals from which antigen is derived. This embodimentof the method of the present invention provides a vaccine whichcompensates for natural variations between individual patients in theexpression of tumor antigens from tumors of the same histological tumortype. Therefore, administration of this therapeutic composition iseffective to elicit an immune response against a variety of tumorantigens such that the same therapeutic composition can be administeredto a variety of different individuals. In some embodiments, antigensfrom tumors of different histological tumor types can be administered toan animal, in order to provide a very broad vaccine.

Preferably, the tumor from which the antigen is isolated or derived isany tumor or cancer, including, but not limited to, melanomas, squamouscell carcinoma, breast cancers, head and neck carcinomas, thyroidcarcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers,prostatic cancers, ovarian cancers, bladder cancers, skin cancers, braincancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primaryhepatic cancers, lung cancers, pancreatic cancers, gastrointestinalcancers, renal cell carcinomas, hematopoietic neoplasias and metastaticcancers thereof. Examples of specific cancer antigens to be used in avaccine of the present invention include but are not limited to, MAGE(including but not limited to MAGE3, MAGEA6, MAGEA10), NY-ESO-1, gp100,tyrosinase, EGF-R, PSA, PMSA, CEA, HER2/neu, Muc-1, hTERT, MART1, TRP-1,TRP-2, BCR-abl, and mutant oncogenic forms of p53 (TP53), p73, ras,BRAF, APC (adenomatous polyposis coli), myc, VHL (von Hippel's Lindauprotein), Rb-1 (retinoblastoma), Rb-2, BRCA1, BRCA2, AR (androgenreceptor), Smad4, MDR1, Flt-3.

According to the present invention, a cancer antigen can include anytumor antigen as described above, in addition to any other antigen thatis associated with the risk of acquiring or development of cancer or forwhich an immune response against such antigen can have a therapeuticbenefit against a cancer. For example, a cancer antigen could include,but is not limited to, a tumor antigen, a mammalian cell moleculeharboring one or more mutated amino acids, a protein normally expressedpre- or neo-natally by mammalian cells, a protein whose expression isinduced by insertion of an epidemiologic agent (e.g. virus), a proteinwhose expression is induced by gene translocation, and a protein whoseexpression is induced by mutation of regulatory sequences. Some of theseantigens may also serve as antigens in other types of diseases (e.g.,autoimmune disease).

In one aspect of the invention, the antigen useful in the presentcomposition is an antigen from a pathogen (including the wholepathogen), and particularly, from a pathogen that is associated with(e.g., causes or contributes to) an infectious disease. An antigen froman infectious disease pathogen can include antigens having epitopes thatare recognized by T cells, antigens having epitopes that are recognizedby B cells, antigens that are exclusively expressed by pathogens, andantigens that are expressed by pathogens and by other cells. Pathogenantigens can include whole cells and the entire pathogen organism, aswell as lysates, extracts or other fractions thereof. In some instances,an antigen can include organisms or portions thereof which may not beordinarily considered to be pathogenic in an animal, but against whichimmunization is nonetheless desired. The antigens can include one, twoor a plurality of antigens that are representative of the substantiallyall of the antigens present in the infectious disease pathogen againstwhich the vaccine is to be administered. In other embodiments, antigensfrom two or more different strains of the same pathogen or fromdifferent pathogens can be used to increase the therapeutic efficacyand/or efficiency of the vaccine.

According to the present invention, a pathogen antigen includes, but isnot limited to, an antigen that is expressed by a bacterium, a virus, aparasite or a fungus. Preferred pathogen antigens for use in the methodof the present invention include antigens which cause a chronicinfectious disease in an animal. In one embodiment, a pathogen antigenfor use in the method or composition of the present invention includesan antigen from a virus. Examples of viral antigens to be used in avaccine of the present invention include, but are not limited to, env,gag, rev, tar, tat, nucleocapsid proteins and reverse transcriptase fromimmunodeficiency viruses (e.g., HIV, FIV); HBV surface antigen and coreantigen; HCV antigens; influenza nucleocapsid proteins; parainfluenzanucleocapsid proteins; human papilloma type 16 E6 and E7 proteins;Epstein-Barr virus LMP-1, LMP-2 and EBNA-2; herpes LAA and glycoproteinD; as well as similar proteins from other viruses. Particularlypreferred antigens for use in the present invention include, but are notlimited to, HIV-1 gag, HIV-1 env, HIV-1 pol, HIV-1 tat, HIV-1 nef,HbsAG, HbcAg, hepatitis c core antigen, HPV E6 and E7, HSV glycoproteinD, and Bacillus anthracis protective antigen.

Other preferred antigens to include in compositions (vaccines) of thepresent invention include antigens that are capable of suppressing anundesired, or harmful, immune response, such as is caused, for example,by allergens, autoimmune antigens, inflammatory agents, antigensinvolved in GVHD, certain cancers, septic shock antigens, and antigensinvolved in transplantation rejection. Such compounds include, but arenot limited to, antihistamines, cyclosporin, corticosteroids, FK506,peptides corresponding to T cell receptors involved in the production ofa harmful immune response, Fas ligands (i.e., compounds that bind to theextracellular or the cytosolic domain of cellular Fas receptors, therebyinducing apoptosis), suitable MHC complexes presented in such a way asto effect tolerization or anergy, T cell receptors, and autoimmuneantigens, preferably in combination with a biological response modifiercapable of enhancing or suppressing cellular and/or humoral immunity.

Other antigens useful in the present invention and combinations ofantigens will be apparent to those of skill in the art. The presentinvention is not restricted to the use of the antigens as describedabove.

According to the present invention, the term “yeast vehicle-antigencomplex” or “yeast-antigen complex” is used generically to describe anyassociation of a yeast vehicle with an antigen. Such associationincludes expression of the antigen by the yeast (a recombinant yeast),introduction of an antigen into a yeast, physical attachment of theantigen to the yeast, and mixing of the yeast and antigen together, suchas in a buffer or other solution or formulation. These types ofcomplexes are described in detail below.

In one embodiment, a yeast cell used to prepare the yeast vehicle istransformed with a heterologous nucleic acid molecule encoding theantigen such that the antigen is expressed by the yeast cell. Such ayeast is also referred to herein as a recombinant yeast or a recombinantyeast vehicle. The yeast cell can then be loaded into the dendritic cellas an intact cell, or the yeast cell can be killed, or it can bederivatized such as by formation of yeast spheroplasts, cytoplasts,ghosts, or subcellular particles, any of which is followed by loading ofthe derivative into the dendritic cell. Yeast spheroplasts can also bedirectly transfected with a recombinant nucleic acid molecule (e.g., thespheroplast is produced from a whole yeast, and then transfected) inorder to produce a recombinant spheroplast that expresses an antigen.

According to the present invention, an isolated nucleic acid molecule ornucleic acid sequence, is a nucleic acid molecule or sequence that hasbeen removed from its natural milieu. As such, “isolated” does notnecessarily reflect the extent to which the nucleic acid molecule hasbeen purified. An isolated nucleic acid molecule useful for transfectingyeast vehicles include DNA, RNA, or derivatives of either DNA or RNA. Anisolated nucleic acid molecule can be double stranded or singlestranded. An isolated nucleic acid molecule useful in the presentinvention includes nucleic acid molecules that encode a protein or afragment thereof, as long as the fragment contains at least one epitopeuseful in a composition of the present invention.

Nucleic acid molecules transformed into yeast vehicles of the presentinvention can include nucleic acid sequences encoding one or moreproteins, or portions thereof. Such nucleic acid molecules can comprisepartial or entire coding regions, regulatory regions, or combinationsthereof. One advantage of yeast strains is their ability to carry anumber of nucleic acid molecules and of being capable of producing anumber of heterologous proteins. A preferred number of antigens to beproduced by a yeast vehicle of the present invention is any number ofantigens that can be reasonably produced by a yeast vehicle, andtypically ranges from at least one to at least about 5 or more, withfrom about 2 to about 5 compounds being more preferred.

A peptide or protein encoded by a nucleic acid molecule within a yeastvehicle can be a full-length protein, or can be a functionallyequivalent protein in which amino acids have been deleted (e.g., atruncated version of the protein), inserted, inverted, substitutedand/or derivatized (e.g., acetylated, glycosylated, phosphorylated,tethered by a glycerophosphatidyl inositol (GPI) anchor) such that themodified protein has a biological function substantially similar to thatof the natural protein (or which has enhanced or inhibited function ascompared to the natural protein, if desired). Modifications can beaccomplished by techniques known in the art including, but not limitedto, direct modifications to the protein or modifications to the nucleicacid sequence encoding the protein using, for example, classic orrecombinant DNA techniques to effect random or targeted mutagenesis.Functionally equivalent proteins can be selected using assays thatmeasure the biological activity of the protein.

Expression of an antigen in a yeast vehicle of the present invention isaccomplished using techniques known to those skilled in the art.Briefly, a nucleic acid molecule encoding at least one desired antigenis inserted into an expression vector in such a manner that the nucleicacid molecule is operatively linked to a transcription control sequencein order to be capable of effecting either constitutive or regulatedexpression of the nucleic acid molecule when transformed into a hostyeast cell. Nucleic acid molecules encoding one or more antigens can beon one or more expression vectors operatively linked to one or moretranscription control sequences.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the yeast cell and thatcontrol the expression of nucleic acid molecules. In particular,recombinant molecules of the present invention include nucleic acidmolecules that are operatively linked to one or more transcriptioncontrol sequences. The phrase “operatively linked” refers to linking anucleic acid molecule to a transcription control sequence in a mannersuch that the molecule is able to be expressed when transfected (i.e.,transformed, transduced or transfected) into a host cell.

Transcription control sequences, which can control the amount of proteinproduced, include sequences which control the initiation, elongation,and termination of transcription. Particularly important transcriptioncontrol sequences are those which control transcription initiation, suchas promoter and upstream activation sequences. Any suitable yeastpromoter can be used in the present invention and a variety of suchpromoters are known to those skilled in the art. Preferred promoters forexpression in Saccharomyces cerevisiae include, but are not limited to,promoters of genes encoding the following yeast proteins: alcoholdehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase(PGK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphatedehydrogenase (GAPDH; also referred to as TDH3, for triose phosphatedehydrogenase), galactokinase (GAL1), galactose-1-phosphateuridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochromec₁ (CYC1), Sec7 protein (SEC7) and acid phosphatase (PHO5), with hybridpromoters such as ADH2/GAPDH and CYC1/GAL10 promoters being morepreferred, and the ADH2/GAPDH promoter, which is induced when glucoseconcentrations in the cell are low (e.g., about 0.1 to about 0.2percent), being even more preferred. Likewise, a number of upstreamactivation sequences (UASs), also referred to as enhancers, are known.Preferred upstream activation sequences for expression in Saccharomycescerevisiae include, but are not limited to, the UASs of genes encodingthe following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1,GAL7 and GAL10, as well as other UASs activated by the GAL4 geneproduct, with the ADH2 UAS being particularly preferred. Since the ADH2UAS is activated by the ADR1 gene product, it is preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Preferred transcription termination sequences forexpression in Saccharomyces cerevisiae include the termination sequencesof the α-factor, GAPDH, and CYC1 genes.

Preferred transcription control sequences to express genes inmethyltrophic yeast include the transcription control regions of thegenes encoding alcohol oxidase and formate dehydrogenase.

Transfection of a nucleic acid molecule into a yeast cell according tothe present invention can be accomplished by any method by which anucleic acid molecule administered into the cell and includes, but isnot limited to, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Transfected nucleic acid molecules can be integrated into ayeast chromosome or maintained on extrachromosomal vectors usingtechniques known to those skilled in the art. Examples of yeast vehiclescarrying such nucleic acid molecules are disclosed in detail herein. Asdiscussed above, yeast cytoplast, yeast ghost, and subcellular yeastmembrane extract or fractions thereof can also be produced recombinantlyby transfecting intact yeast microorganisms or yeast spheroplasts withdesired nucleic acid molecules, producing the antigen therein, and thenfurther manipulating the microorganisms or spheroplasts using techniquesknown to those skilled in the art to produce cytoplast, ghost orsubcellular yeast membrane extract or fractions thereof containingdesired antigens.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen by the yeast vehicle include an effectivemedium in which a yeast strain can be cultured. An effective medium istypically an aqueous medium comprising assimilable carbohydrate,nitrogen and phosphate sources, as well as appropriate salts, minerals,metals and other nutrients, such as vitamins and growth factors. Themedium may comprise complex nutrients or may be a defined minimalmedium. Yeast strains of the present invention can be cultured in avariety of containers, including, but not limited to, bioreactors,erlenmeyer flasks, test tubes, microtiter dishes, and petri plates.Culturing is carried out at a temperature, pH and oxygen contentappropriate for the yeast strain. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art (see, forexample, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194,Academic Press, San Diego).

In one embodiment of the present invention, as an alternative toexpression of an antigen recombinantly in the yeast vehicle, a yeastvehicle is loaded intracellularly with the protein or peptide antigen,or with carbohydrates or other molecules that serve as an antigen.Subsequently, the yeast vehicle, which now contains the antigenintracellularly, can be administered to the patient or loaded into acarrier such as a dendritic cell (described below). As used herein, apeptide comprises an amino acid sequence of less than or equal to about30-50 amino acids, while a protein comprises an amino acid sequence ofmore than about 30-50 amino acids; proteins can be multimeric. A proteinor peptide useful as an antigen can be as small as a T cell epitope(i.e., greater than 5 amino acids in length) and any suitable sizegreater than that which comprises multiple epitopes, protein fragments,full-length proteins, chimeric proteins or fusion proteins. Peptides andproteins can be derivatized either naturally or synthetically; suchmodifications can include, but are not limited to, glycosylation,phosphorylation, acetylation, myristylation, prenylation,palmitoylation, amidation and/or addition of glycerophosphatidylinositol. Peptides and proteins can be inserted directly into yeastvehicles of the present invention by techniques known to those skilledin the art, such as by diffusion, active transport, liposome fusion,electroporation, phagocytosis, freeze-thaw cycles and bath sonication.Yeast vehicles that can be directly loaded with peptides, proteins,carbohydrates, or other molecules include intact yeast, as well asspheroplasts, ghosts or cytoplasts, which can be loaded with antigensafter production, but before loading into dendritic cells.Alternatively, intact yeast can be loaded with the antigen, and thenspheroplasts, ghosts, cytoplasts, or subcellular particles can beprepared therefrom. Any number of antigens can be loaded into a yeastvehicle in this embodiment, from at least 1, 2, 3, 4 or any wholeinteger up to hundreds or thousands of antigens, such as would beprovided by the loading of a microorganism, by the loading of amammalian tumor cell, or portions thereof, for example.

In another embodiment of the present invention, an antigen is physicallyattached to the yeast vehicle. Physical attachment of the antigen to theyeast vehicle can be accomplished by any method suitable in the art,including covalent and non-covalent association methods which include,but are not limited to, chemically crosslinking the antigen to the outersurface of the yeast vehicle or biologically linking the antigen to theouter surface of the yeast vehicle, such as by using an antibody orother binding partner. Chemical cross-linking can be achieved, forexample, by methods including glutaraldehyde linkage, photoaffinitylabeling, treatment with carbodiimides, treatment with chemicals capableof linking di-sulfide bonds, and treatment with other cross-linkingchemicals standard in the art. Alternatively, a chemical can becontacted with the yeast vehicle that alters the charge of the lipidbilayer of yeast membrane or the composition of the cell wall so thatthe outer surface of the yeast is more likely to fuse or bind toantigens having particular charge characteristics. Targeting agents suchas antibodies, binding peptides, soluble receptors, and other ligandsmay also be incorporated into an antigen as a fusion protein orotherwise associated with an antigen for binding of the antigen to theyeast vehicle.

In yet another embodiment, the yeast vehicle and the antigen areassociated with each other by a more passive, non-specific ornon-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen together in a buffer or other suitableformulation. In one embodiment of the invention, the yeast vehicle andthe antigen are both loaded intracellularly into a carrier such as adendritic cell or macrophage to form the therapeutic composition orvaccine of the present invention. Various forms in which the loading ofboth components can be accomplished are discussed in detail below. Asused herein, the term “loaded” and derivatives thereof refer to theinsertion, introduction, or entry of a component (e.g., the yeastvehicle and/or antigen) into a cell (e.g., a dendritic cell). To load acomponent intracellularly refers to the insertion or introduction of thecomponent to an intracellular compartment of the cell (e.g., through theplasma membrane and at a minimum, into the cytoplasm, a phagosome, alysosome, or some intracellular space of the cell). To load a componentinto a cell references any technique by which the component is eitherforced to enter the cell (e.g., by electroporation) or is placed in anenvironment (e.g., in contact with or near to a cell) where thecomponent will be substantially likely to enter the cell by some process(e.g., phagocytosis). Loading techniques include, but are not limitedto: diffusion, active transport, liposome fusion, electroporation,phagocytosis, and bath sonication. In a preferred embodiment, passivemechanisms for loading a dendritic cell with the yeast vehicle and/orantigen are used, such passive mechanisms including phagocytosis of theyeast vehicle and/or antigen by the dendritic cell.

In one embodiment of the present invention, a composition of vaccine canalso include biological response modifier compounds, or the ability toproduce such modifiers (i.e., by transfection with nucleic acidmolecules encoding such modifiers), although such modifiers are notnecessary to achieve a robust immune response according to theinvention. For example, a yeast vehicle can be transfected with orloaded with at least one antigen and at least one biological responsemodifier compound. Biological response modifiers are compounds that canmodulate immune responses. Certain biological response modifiers canstimulate a protective immune response whereas others can suppress aharmful immune response. Certain biological response modifierspreferentially enhance a cell-mediated immune response whereas otherspreferentially enhance a humoral immune response (i.e., can stimulate animmune response in which there is an increased level of cellularcompared to humoral immunity, or vice versa). There are a number oftechniques known to those skilled in the art to measure stimulation orsuppression of immune responses, as well as to differentiate cellularimmune responses from humoral immune responses.

Suitable biological response modifiers include cytokines, hormones,lipidic derivatives, small molecule drugs and other growth modulators,such as, but not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4),interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma(IFN-gamma) insulin-like growth factor I (IGF-I), transforming growthfactor beta (TGF-β) steroids, prostaglandins and leukotrienes. Theability of a yeast vehicle to express (i.e., produce), and possiblysecrete, IL-2, IL-12 and/or IFN-gamma preferentially enhancescell-mediated immunity, whereas the ability of a yeast vehicle toexpress, and possibly secrete, IL-4, IL-5 and/or IL-10 preferentiallyenhances humoral immunity.

Yeast vehicles of the present invention can be associated with a widevariety of antigens capable of protecting an animal from disease, andthis ability can be further enhanced by loading the yeast vehicle andantigen into a dendritic cell or macrophage to form a vaccine of thepresent invention. Accordingly, the method of use of the therapeuticcomposition or vaccine of the present invention preferably elicits animmune response in an animal such that the animal is protected from adisease that is amenable to elicitation of an immune response, includingcancer or an infectious disease. As used herein, the phrase “protectedfrom a disease” refers to reducing the symptoms of the disease; reducingthe occurrence of the disease, and/or reducing the severity of thedisease. Protecting an animal can refer to the ability of a therapeuticcomposition of the present invention, when administered to an animal, toprevent a disease from occurring and/or to cure or to alleviate diseasesymptoms, signs or causes. As such, to protect an animal from a diseaseincludes both preventing disease occurrence (prophylactic treatment orprophylactic vaccine) and treating an animal that has a disease or thatis experiencing initial symptoms of a disease (therapeutic treatment ora therapeutic vaccine). In particular, protecting an animal from adisease is accomplished by eliciting an immune response in the animal byinducing a beneficial or protective immune response which may, in someinstances, additionally suppress (e.g., reduce, inhibit or block) anoveractive or harmful immune response. The term, “disease” refers to anydeviation from the normal health of an animal and includes a state whendisease symptoms are present, as well as conditions in which a deviation(e.g., infection, gene mutation, genetic defect, etc.) has occurred, butsymptoms are not yet manifested.

More specifically, a vaccine as described herein, when administered toan animal by the method of the present invention, preferably produces aresult which can include alleviation of the disease (e.g., reduction ofat least one symptom or clinical manifestation of the disease),elimination of the disease, reduction of a tumor or lesion associatedwith the disease, elimination of a tumor or lesion associated with thedisease, prevention or alleviation of a secondary disease resulting fromthe occurrence of a primary disease (e.g., metastatic cancer resultingfrom a primary cancer), prevention of the disease, and stimulation ofeffector cell immunity against the disease.

Cancers to be treated or prevented using the method and composition ofthe present invention include, but are not limited to, melanomas,squamous cell carcinoma, breast cancers, head and neck carcinomas,thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicularcancers, prostatic cancers, ovarian cancers, bladder cancers, skincancers, brain cancers, angiosarcomas, hemangiosarcomas, mast celltumors, primary hepatic cancers, lung cancers, pancreatic cancers,gastrointestinal cancers, renal cell carcinomas, hematopoieticneoplasias, and metastatic cancers thereof. Particularly preferredcancers to treat with a therapeutic composition of the present inventioninclude primary lung cancers, pulmonary metastatic cancers, primarybrain cancers, and metastatic brain cancers. A preferred brain cancer totreat includes, but is not limited to, glioblastoma multiforme.Preferred lung cancers to treat include, but are not limited to,non-small cell carcinomas, small cell carcinomas and adenocarcinomas. Atherapeutic composition of the present invention is useful for elicitingan immune response in an animal to treat tumors that can form in suchcancers, including malignant and benign tumors. Preferably, expressionof the tumor antigen in a tissue of an animal that has cancer produces aresult selected from the group of alleviation of the cancer, reductionof a tumor associated with the cancer, elimination of a tumor associatedwith the cancer, prevention of metastatic cancer, prevention of thecancer and stimulation of effector cell immunity against the cancer.

One particular advantage of the present invention is that thetherapeutic composition does not need to be administrated with animmunopotentiator such as an adjuvant or a carrier, since the yeastvehicle and antigen combination elicits a potent immune response in theabsence of additional adjuvants, which is again enhanced by loading ofthese components into a dendritic cell, as described in U.S. applicationSer. No. 09/991,363, supra. This characteristic, however, does notpreclude the use of immunopotentiators in compositions of the presentinvention. As such, in one embodiment, a composition of the presentinvention can include one or more adjuvants and/or carriers.

Adjuvants are typically substances that generally enhance the immuneresponse of an animal to a specific antigen. Suitable adjuvants include,but are not limited to, Freund's adjuvant; other bacterial cell wallcomponents; aluminum-based salts; calcium-based salts; silica;polynucleotides; toxoids; serum proteins; viral coat proteins; otherbacterial-derived preparations; gamma interferon; block copolymeradjuvants, such as Hunter's Titermax adjuvant (CytRx™, Inc. Norcross,Ga.); Ribi adjuvants (available from Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives, such as Quil A(available from Superfos Biosector A/S, Denmark).

Carriers are typically compounds that increase the half-life of atherapeutic composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled releaseformulations, biodegradable implants, liposomes, oils, esters, andglycols.

Therapeutic compositions of the present invention can also contain oneor more pharmaceutically acceptable excipients. As used herein, apharmaceutically acceptable excipient refers to any substance suitablefor delivering a therapeutic composition useful in the method of thepresent invention to a suitable in vivo or ex vivo site. Preferredpharmaceutically acceptable excipients are capable of maintaining ayeast vehicle (or a dendritic cell comprising the yeast vehicle) in aform that, upon arrival of the yeast vehicle or cell at a target cell,tissue, or site in the body, the yeast vehicle (associated with anantigen) or the dendritic cell (loaded with a yeast vehicle andantigen), is capable of eliciting an immune response at the target site(noting that the target site can be systemic). Suitable excipients ofthe present invention include excipients or formularies that transport,but do not specifically target the vaccine to a site (also referred toherein as non-targeting carriers). Examples of pharmaceuticallyacceptable excipients include, but are not limited to water, saline,phosphate buffered saline, Ringer's solution, dextrose solution,serum-containing solutions, Hank's solution, other aqueousphysiologically balanced solutions, oils, esters and glycols. Aqueouscarriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal, m- or o-cresol, formalin and benzol alcohol.

The present invention includes the delivery of a composition or vaccineof the invention to an animal. The administration process can beperformed ex vivo or in vivo. Ex vivo administration refers toperforming part of the regulatory step outside of the patient, such asadministering a composition of the present invention to a population ofcells (dendritic cells) removed from a patient under conditions suchthat the yeast vehicle and antigen are loaded into the cell, andreturning the cells to the patient. The therapeutic composition of thepresent invention can be returned to a patient, or administered to apatient, by any suitable mode of administration.

Administration of a vaccine or composition, including a dendritic cellloaded with the yeast vehicle and antigen, can be systemic, mucosaland/or proximal to the location of the target site (e.g., near a tumor).The preferred routes of administration will be apparent to those ofskill in the art, depending on the type of condition to be prevented ortreated, the antigen used, and/or the target cell population or tissue.Preferred methods of administration include, but are not limited to,intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, subcutaneous administration,intraarticular administration, intraventricular administration,inhalation (e.g., aerosol), intracranial, intraspinal, intraocular,aural, intranasal, oral, pulmonary administration, impregnation of acatheter, and direct injection into a tissue. Particularly preferredroutes of administration include: intravenous, intraperitoneal,subcutaneous, intradermal, intranodal, intramuscular, transdermal,inhaled, intranasal, oral, intraocular, intraarticular, intracranial,and intraspinal. Parenteral delivery can include intradermal,intramuscular, intraperitoneal, intrapleural, intrapulmonary,intravenous, subcutaneous, atrial catheter and venal catheter routes.Aural delivery can include ear drops, intranasal delivery can includenose drops or intranasal injection, and intraocular delivery can includeeye drops. Aerosol (inhalation) delivery can also be performed usingmethods standard in the art (see, for example, Stribling et al., Proc.Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated hereinby reference in its entirety). For example, in one embodiment, acomposition or vaccine of the invention can be formulated into acomposition suitable for nebulized delivery using a suitable inhalationdevice or nebulizer. Oral delivery can include solids and liquids thatcan be taken through the mouth, and is useful in the development ofmucosal immunity and since compositions comprising yeast vehicles can beeasily prepared for oral delivery, for example, as tablets or capsules,as well as being formulated into food and beverage products. Otherroutes of administration that modulate mucosal immunity are useful inthe treatment of viral infections, epithelial cancers, immunosuppressivedisorders and other diseases affecting the epithelial region. Suchroutes include bronchial, intradermal, intramuscular, intranasal, otherinhalatory, rectal, subcutaneous, topical, transdermal, vaginal andurethral routes.

A more preferred route of delivery is any route of delivery of acomposition or vaccine to the respiratory system, including, but notlimited to, inhalation, intranasal, intratracheal, and the like. Asdiscussed above and shown in the Examples, the present inventors haveshown that administration of a vaccine of the invention by this route ofadministration provides enhanced results as compared to at leastsubcutaneous delivery, and appears to be particularly efficacious forthe treatment of brain cancers and lung cancers.

According to the present invention, an effective administration protocol(i.e., administering a vaccine or therapeutic composition in aneffective manner) comprises suitable dose parameters and modes ofadministration that result in elicitation of an immune response in ananimal that has a disease or condition, or that is at risk ofcontracting a disease or condition, preferably so that the animal isprotected from the disease. Effective dose parameters can be determinedusing methods standard in the art for a particular disease. Such methodsinclude, for example, determination of survival rates, side effects(i.e., toxicity) and progression or regression of disease. Inparticular, the effectiveness of dose parameters of a therapeuticcomposition of the present invention when treating cancer can bedetermined by assessing response rates. Such response rates refer to thepercentage of treated patients in a population of patients that respondwith either partial or complete remission. Remission can be determinedby, for example, measuring tumor size or microscopic examination for thepresence of cancer cells in a tissue sample.

In accordance with the present invention, a suitable single dose size isa dose that is capable of eliciting an antigen-specific immune responsein an animal when administered one or more times over a suitable timeperiod. Doses can vary depending upon the disease or condition beingtreated. In the treatment of cancer, for example, a suitable single dosecan be dependent upon whether the cancer being treated is a primarytumor or a metastatic form of cancer. One of skill in the art canreadily determine appropriate single dose sizes for administration basedon the size of an animal and the route of administration.

A suitable single dose of a therapeutic composition or vaccine of thepresent invention is a dose that is capable of effectively providing ayeast vehicle and an antigen to a given cell type, tissue, or region ofthe patient body in an amount effective to elicit an antigen-specificimmune response, when administered one or more times over a suitabletime period. For example, in one embodiment, a single dose of a yeastvehicle of the present invention is from about 1×10⁵ to about 5×10⁷yeast cell equivalents per kilogram body weight of the organism beingadministered the composition. More preferably, a single dose of a yeastvehicle of the present invention is from about 0.1 Y.U. (1×10⁶ cells) toabout 100 Y.U. (1×10⁹ cells) per dose (i.e., per organism), includingany interim dose, in increments of 0.1×10⁶ cells (i.e., 1.1×10⁶,1.2×10⁶, 1.3×10⁶ . . . ). This range of doses can be effectively used inany organism of any size, including mice, monkeys, humans, etc. When thevaccine is administered by loading the yeast vehicle and antigen intodendritic cells, a preferred single dose of a vaccine of the presentinvention is from about 0.5×10⁶ to about 40×10⁶ dendritic cells perindividual per administration. Preferably, a single dose is from about1×10⁶ to about 20×10⁶ dendritic cells per individual, and morepreferably from about 1×10⁶ to about 10×10⁶ dendritic cells perindividual. “Boosters” of a therapeutic composition are preferablyadministered when the immune response against the antigen has waned oras needed to provide an immune response or induce a memory responseagainst a particular antigen or antigen(s). Boosters can be administeredfrom about 2 weeks to several years after the original administration.In one embodiment, an administration schedule is one in which from about1×10⁵ to about 5×10⁷ yeast cell equivalents of a composition per kg bodyweight of the organism is administered from about one to about 4 timesover a time period of from about 1 month to about 6 months.

It will be obvious to one of skill in the art that the number of dosesadministered to an animal is dependent upon the extent of the diseaseand the response of an individual patient to the treatment. For example,a large tumor may require more doses than a smaller tumor, and a chronicdisease may require more doses than an acute disease. In some cases,however, a patient having a large tumor may require fewer doses than apatient with a smaller tumor, if the patient with the large tumorresponds more favorably to the therapeutic composition than the patientwith the smaller tumor. Thus, it is within the scope of the presentinvention that a suitable number of doses includes any number requiredto treat a given disease. In another aspect of the invention, the methodof treatment of a disease or condition such as cancer can be combinedwith other therapeutic approaches to enhance the efficacy of thetreatment. For example, in the treatment of cancer, the administrationof the vaccine of the present invention can occur after surgicalresection of a tumor from the animal. In another aspect, administrationof the vaccine occurs after surgical resection of a tumor from theanimal and after administration of non-myeloablative allogeneic stemcell transplantation (discussed below). In yet another aspect,administration of the vaccine occurs after surgical resection of a tumorfrom the animal, after administration of non-myeloablative allogeneicstem cell transplantation, and after allogeneic donor lymphocyteinfusion.

Another embodiment of the present invention relates to a method to treata patient that has cancer, comprising: (a) treating a patient that hascancer by nonmyeloablative stem cell transfer effective to establish astable mixed bone marrow chimerism, wherein the stem cells are providedby an allogeneic donor; (b) administering lymphocytes obtained from theallogeneic donor to the patient; and (c) administering to the patient,after step (b), a vaccine comprising a yeast vehicle and at least onecancer antigen. The process of establishing a stable mixed bone marrowchimerism via non-myeloablative allogeneic stem cell transplantation hasbeen previously described in detail in Luznik et al. (Blood 101(4):1645-1652, 2003) and elsewhere in the art (e.g., Appelbaum et al., 2001,Hematology pp. 62-86). Briefly, a patient is treated with non-lethal,non-myeloablative total body irradiation and immunosuppression (e.g.,combination radiation and chemotherapy) and is administered a populationof cells containing stem cells (e.g., bone marrow) from an allogeneicdonor. This treatment will result in the establishment of stable, mixedbone marrow chimerism in the recipient patient (i.e., both donor andhost immune cells exist). In the protocol of Luznik et al., therecipient is then provided with an infusion of donor lymphocytes,followed by a vaccine of autologous tumor cells, a source of GM-CSF anda source of histocompatibility antigens. This treatment resulted in longterm tumor free survival of a significant number of the experimentalanimals.

The present invention provides an improvement to the non-myeloablativeallogeneic stem cell transplantation and tumor cell vaccination protocolby combining the non-myeloablative allogeneic stem cell transplantationwith a yeast-based vaccine strategy of the present invention. Asexemplified in Example 5, the method of the present invention is aseffective at treating tumors as the protocol of Luznik et al., but doesnot require the use of autologous tumor antigens from the recipient, northe use of biological response modifiers or other adjuvants (e.g., theGM-CSF and source of histocompatibility antigens) as provided in theprior protocol. The modified method of the present invention providesadditional advantages of enabling the use of a wide variety of veryspecific antigen selections and combinations in the vaccine, and ofproviding a vaccine for a broad spectrum of cancer patients, whereas theprior protocol, by utilizing autologous tumor cells from the recipient,is effectively limited to that patient. The present invention alsoprovides for the vaccination of the donor of stem cells and lymphocyteswith the yeast-based vaccine of the invention, which can express thesame or slightly different antigens as the vaccine to be administered tothe recipient, which is expected to further enhance the efficacy of thevaccine.

In this embodiment of the invention, the step of treating a patient thathas cancer by nonmyeloablative stem cell transfer effective to establisha stable mixed bone marrow chimerism, wherein the stem cells areprovided by an allogeneic donor is performed as has been well describedin the art (e.g., Luznik et al., supra; Appelbaum et al., 2001,Hematology pp. 62-86). The allogeneic lymphocyte infusion of step (b)can be performed by any suitable method, including collection ofallogeneic lymphocytes from peripheral blood of the donor and infusioninto the recipient patient, such as by Ultrapheresis techniques known inthe art. Finally, the patient is administered the yeast-based vaccine ofthe invention as previously described herein. In one aspect of thisembodiment, the method further includes administering to the donor,prior to step (a), a vaccine comprising a yeast vehicle and at least onecancer antigen. In another aspect, the method includes removing a tumorfrom the patient prior to performing step (a).

In the method of the present invention, vaccines and therapeuticcompositions can be administered to any member of the Vertebrate class,Mammalia, including, without limitation, primates, rodents, livestockand domestic pets. Livestock include mammals to be consumed or thatproduce useful products (e.g., sheep for wool production). Preferredmammals to protect include humans, dogs, cats, mice, rats, goats, sheep,cattle, horses and pigs, with humans being particularly preferred.According to the present invention, the term “patient” can be used todescribe any animal that is the subject of a diagnostic, prophylactic,or therapeutic treatment as described herein.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example demonstrates the administration of a yeast basedvaccine comprising a cancer antigen for the treatment of a non-smallcell lung carcinoma (NSCLC) in vivo.

Ras mutations are common in pulmonary adenocarcinomas of humans, mice,rats and hamsters. In fact, mutations in the ras proto-oncogene familyare the most common oncogene-related mutations in human cancer and intumors in experimental animals. The present inventors tested whetheryeast-based vaccines which have now been designed to be directed tomutant protein-specific ras mutations, can induce productive immuneresponses that lead to tumor destruction in mouse lung adenocarcinomamodels. The overall goal of the experiments was to establish that such avaccine could be used to combat lung cancer in humans.

The model used in the experiments described herein is a mouse model inwhich A/J mice are injected with urethane (ethyl carbamate, which ismetabolized to vinyl carbamate, the presumptive carcinogenicmetabolite). Hyperplasias are seen in about 6 weeks, benign tumors at8-10 weeks with the first signs of malignancy after 8 months. By 10months the tumors can occupy the whole lung lobe and at 12 months themice die from respiratory distress. In this experiment, a single K-rasmutation is expressed in the tumor cells, which is in the codon encodingthe amino acid residue at position 61 (also referred to as codon 61).

The present inventors have produced Ras61-VAX (GlobeImmune), which is astrain of yeast that has been engineered to expresses mouse K-rasprotein with a mutation at codon 61 (relative to the K-ras sequence ofSEQ ID NO:5), which is the mutant K-ras protein expressed inspontaneously induced mouse lung tumors and mouse lung tumor cell lines.Animals immunized with the Ras61-VAX directed against codon 61 mutationswere tested for their ability to prevent the development of tumors orreduce their size after induction in the urethane induction model.

The results demonstrated that animals immunized with Ras61-VAX showsignificant protection against pre-existing lung tumors spontaneouslyinduced by urethane exposure in mice. Both the number of tumors and thesize of tumors was significantly reduced in vaccinated animals, comparedto control animals (FIG. 1). These results demonstrate the feasibilityand utility of therapeutic intervention using the present inventors'yeast-based vaccines that express mutant K-ras proteins to treat and/orprevent disease caused by a cancer.

In addition, FIG. 2 shows the results of an experiment in which C57BL/6mice were immunized by subcutaneous administration of Ras61-VAX (Q61Ralone) or by intranasal versus subcutaneous administration of a yeastvaccine expressing a mutant Ras having two mutations (RasV-VAX;G12V+Q61R), on days 1, 8, 22 and 36. Mice were challenged with 10,000CMT64 cells by subcutaneous administration on day 29, where CMT64 cellsendogenously express a mutant K-ras protein altered at amino acid 12from glycine to valine (G12V). FIG. 2 shows the size of tumors on day 59(30 days after challenge) and the number of animals with tumors/totalnumber of animals (above bar). As shown in FIG. 2, administration of theRas61-VAX again provided minimal protection against lung tumor growth (2out of 7 animals are tumor-free), and administration of RasV-Vaxprovided specific immunotherapeutic protection by significantly reducingtumor volume and numbers (4 out of 8 animals vaccinated subcutaneouslyare tumor-free and 7 out of 8 animals vaccinated intranasally aretumor-free). Surprisingly, intranasal administration of the vaccineprovided superior results as compared to the subcutaneous administrationof the same vaccine. These results highlighted the specificity ofmolecular immunotherapy with the yeast-based vaccine products. Thesestudies revealed the requirement that immune-mediated rejection of tumorgrowth was dependent on the administration of yeast-based vaccines withthe tumor antigen harboring the relevant mutated amino acid.

Example 2

The following example demonstrates the use of a yeast-based vaccinecomprising a cancer antigen to treat a brain tumor in vivo.

In the following experiment, groups of 5 mice were immunized twice (day0 and day 7) with Gag protein-expressing vaccine (GI-VAX) or PBS (mockinjected) by subcutaneous injection or intranasal administration, thenchallenged on day 14 with tumors expressing the Gag protein. The resultsfrom two independent studies revealed prolonged survival againstintracranial tumor challenge in mice receiving the vaccine by intranasaladministration, as compared to mock-injected mice, and surprisingly, ascompared to animals receiving the vaccine by a subcutaneous route (FIG.3). Subcutaneous immunization did protect animals against subcutaneoustumor challenge (data not shown). These results show that the method ofthe present invention can be used effectively when administeredintranasally and that administration to the respiratory tract may beefficacious for intracranial tumors where other routes of administrationare not.

Example 3

The following example demonstrates the use of a yeast-based vaccinecomprising a human cancer antigen (epidermal growth factor receptor;EGFR) to treat a melanoma and a brain tumor in vivo.

The ability of immunotherapeutic strategies to elicit protective immuneresponses is dependent on a number of important variables. First, thevaccine must be able to activate the immune system to recognize thetarget antigen, i.e. to provide “adjuvant” activity. In the case of theyeast-based vaccine, the inventors had previously shown that uptake ofyeast into dendritic cells upregulated MHC class I and class II proteinexpression, and to trigger cytokine production, which are the hallmarksof adjuvant activity (Stubbs et al, Nature Med (2001) 7, 625-629). Thedegree to which yeast activate the ‘innate’ immune system was equivalentto that seen by using lipopolysaccharide (LPS) derived from bacterialcell walls. Second, the vaccine must promote surface presentation of theimmunodominant epitopes of the target antigens to the immune system. Theinventors had previously demonstrated that the yeast-based vaccine isvery potent for delivering antigenic epitopes for stimulation of thecell-mediated (CTL) and the humoral (antibody) responses of the immunesystem (Stubbs et al, Nature Medicine (2001) 7, 625-629). Third, andmost importantly, stimulation of the immune system must trigger immuneresponses to sites in the body where they are needed. As shown below,surprisingly, the route of vaccine administration appears to influencethe efficacy of the immune response against tumors that develop indifferent sites in the body.

To test the immunogenicity of an EGFR-tm VAX (a yeast vaccine of theinvention expressing EGFR as the cancer antigen), it was necessary tomodify the glioma tumor cells used in the challenge experiments. B16mouse melanoma cells and 9L rat glioma tumor cells were transfected toexpress human EGFR (B16-E cells and 9L-E cells, respectively). Thecloned 9L-E cell line was subsequently sorted for cells that expresshigh, intermediate or low levels of hEGFR. The B16-E cells and the 9L-Ecells therefore possess the antigen included in the yeast vaccine (i.e.human EGFR), and provide an appropriate surrogate model for humangliomas that exhibit altered expression of EGFR in the malignant cells.The goal of the studies was to demonstrate that the yeast-based deliveryvehicle triggered protective immunity against challenge with a lethaldose of the 9L-E glioma cells implanted intracranially into rats.

The B16-E cells and 9L-E cells were cloned to homogeneity and shown toexpress human EGFR, as assayed by flow cytometry. To ensure that theheterologous expression of the human EGFR protein did not result inimmune rejection of the tumors in the absence of vaccine administration,the transfected B16-E were first determined to be capable of formingsubcutaneous tumors in mice (data not shown). The transfected 9L-E cellsformed tumors subcutaneously and intracranially in rats (data notshown). Now the stage was set for testing the efficacy of EGFR-tm VAXyeast vaccine for protecting animals against B16-E tumor challenge inmice and 9L-E tumor challenge in rats.

Preliminary vaccine challenge studies were designed to determine whethersubcutaneous vaccination with EGFR-tm VAX is efficacious for protectinganimals against challenge with a lethal dose of the B16-E melanoma tumorcells implanted subcutaneously. This approach represents one of theinventors' standard measure for the utility of a new target tumorantigen to be effective for eliciting tumor cell killing. This studydemonstrated that animals vaccinated with EGFR-tm VAX are protectedagainst B16-E tumor challenge (4/6 animals are tumor-free), as comparedto mock-immunized animals (1/6 animals are tumor-free) (data not shown).These results validate that EGFR serves as an appropriate antigen foreliciting cell-mediated immune responses, and that the EGFR-tm vaccinetriggers protective immune responses against tumor challenge. Therefore,the next step was to test the efficacy of EGFR-tm VAX againstintracranial challenge with 9L-E gliomas in rats.

The inventors also demonstrated in the experiment above that theyeast-based vaccine, when administered intranasally (i.n.), providesequivalent protection as subcutaneous immunization of the vaccineagainst subcutaneous melanoma tumor challenge (data not shown).Therefore, the next experiment tested whether the yeast-basedimmunotherapeutic EGFR-VAX product, which was demonstrated to elicitprotective immune responses against a subcutaneous B16 melanoma tumorchallenge, would provide immunotherapeutic protection against anintracranial tumor challenge.

The efficacy of the EGFR-tm VAX and the impact of route ofadministration was further tested by intracranial challenge with gliomatumor cells in the rat model. Animals (8 animals per group) wereimmunized with 20 million yeast cells expressing hEGFR (EGFR-vax) oryeast (vector alone) by the intranasal (i.n.) or subcutaneous (s.c.)route on days 0, 7, 21. Immunized animals were challenged byintracranial administration of 1,250 cells of the untransfected 9L ratglioma (9L alone) or 9L expressing hEGFR. Rat body weights weremonitored daily, where loss of body weight was indicative of impendinganimal mortality.

The results (FIG. 4) demonstrated that 50% of the animals immunized withEGFR-VAX yeast were completely protected against lethal intracranialtumor challenge with the rat 9L glioma expressing the tumor antigen, butnone of the animals rejected the growth of tumors that lack the tumorantigen (i.e., the vaccine induces antigen-specific immunity). Inaddition, the remaining EGFR-VAX-immunized animals that succumbed to thelethal challenge still demonstrated extended survival time as comparedto control animals.

Furthermore, the statistically significant improvement in survival ofanimals that were immunized intranasally as compared to subcutaneouslyis both intriguing and surprising, and reproduces the data that weredescribed above (see Example 2) regarding protection againstintracranial (melanoma) tumor challenge in mice.

Because this rat intracranial tumor challenge model is considered tomost closely reflect human glioma, positive data with these studiesprovide excellent pre-clinical data for moving into a clinical trial.Additional studies can include dose ranging, schedule, surgicalre-section studies, and re-challenge of 9L-E survivors with 9L tumors toexamine whether the immune system is now “educated” with regard toadditional (unknown) tumor antigens in 9L gliomas, as well as testing ofyeast vehicles expressing the EGFR-vIII mutant protein, and willestablish a basis to begin manufacturing of clinical grade vaccineproduct.

The data described above indicate that while multiple routes ofimmunization may be effective for destroying tumors in the periphery,the yeast-based vaccines of the present invention are particularlyefficacious for priming effector cells that may be unique to the lung.Since the yeast-based vaccine can prime unique effector cell precursors,the immune cells activated by intranasal immunization may beparticularly effective for crossing the blood-brain barrier to influencethe course of intracranial tumor growth. Therefore, the route ofimmunization may be a critical and previously unappreciated component inthe design of an effective yeast-based vaccine for brain tumors. Becausethe yeast-based vaccine is extremely facile for multiple routes ofimmunization the vaccine holds the promise to uniquely provoke highlyspecialized immune responses with heretofore underappreciated potentialfor the treatment of some cancers.

Example 4

The following example demonstrates the use of a yeast-based vaccinecomprising a cancer antigen to treat renal cancer in vivo.

In 2001, renal cell cancer (RCC) will be diagnosed in approximately31,800 individuals in the United States, with 11,600 deaths; thisrepresents 2 to 3 percent of all cancers and 2 percent of all deathsfrom neoplasms. Although patients traditionally presented with the triadof hematuria, abdominal mass, pain, and weight loss, fewer currentlydiagnosed patients have these symptoms because of the increasedfrequency of incidental diagnosis. Many patients are diagnosed withdisease that, although potentially curable by surgery, will relapsebecause cells have already reached the vascular system. Moreover,therapy for metastatic RCC is extremely limited. Hormonal andchemotherapeutic approaches produce <10% response rates and noappreciable change in survival. However, there has been a long-standinginterest in the use of immunologic treatment for the disease. Inaddition to the rare instances of spontaneous regression, bothα-interferon and interleukin-2 have shown “significant” activity with adefinite minority of patients responding to treatment, some withcomplete remissions. Although there are few prospective randomizedtrials, a recent abstract from the Cytokine Working Group documented an8% complete response rate and 25% overall response rate to high-doseIL-2 compared with about half the response rate with outpatientsubcutaneous IL-2/α-interferon. Overall, while clearly showing activityagainst RCC, approaches used to date have lacked both specificity forthe disease and potency.

Over 60% of RCCs carry inactivating mutations in VHL, which appears toact as a “gatekeeper” gene for RCC, analogous to the role of APC incolon cancer. The protein encoded by VHL is an essential component of anE3 ubiquitin-ligation (SCF) complex, known as VHL/elonginCB/Cul-2 (VCB),which targets particular proteins for destruction by the 26S proteasome.Since many VHL mutations result in missense or frameshifted proteins,novel epitopes will be generated that should be recognized astumor-specific antigens. The following experiments tested the hypothesisthat mutant VHL proteins in RCCs can be targeted for immune responsesafter incorporation into a novel yeast-based vaccine of the presentinvention.

There are no comparable mutated VHL mediated tumors in mice. Therefore,the present inventors used the known human VHL sequence (SEQ ID NO:16)as well as cloned mouse VHL (SEQ ID NO:17) to prepare expressionconstructs encoding murine VHL sequences which are either wild-type orcarry two specific mutations affecting Y98 or R167 (with respect to themurine sequence of SEQ ID NO: 17). Mutations in these two positionscorrespond to hot spots frequently found in human tumors. Tyrosine 98forms a surface exposed binding site for VHL targets such as HIF1α whilearginine 167 is important for stabilization of the alpha helix H1. Bothof these residues are significantly exposed to solvent and are likely tobe accessible for immune system recognition. As shown in the BLASTcomparison below, human and murine VHL amino acid sequences are nearlyidentical from position 58 through 190, including these two hot spots.

     58                                 Tyrosine98            117 hVHL:RPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLW SEQ ID NO:16 mVHL: RPRPVLRSVNSREPSQVIFCNRSPRVVLPLWLNFDGEPQPYPILPPGTGRRIHSYRGHLWSEQ ID NO: 17     24                                                        83     118                                       Arginine167    177 hVHL:LFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRR mVHL:LFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRR     84                                                       143     178                            211 hVHL:LDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRM mVHL:LDIVRSLYEDLEDYPSVRKDIQRLSQEHLESQHL     144                            177

Therefore, results obtained with these murine constructs provide areasonably accurate estimate of the effectiveness in human RCC. Y98 ismost frequently mutated into histidine, while R167 is typically mutatedto glutamine or tryptophane. R167 is also affected by frame shiftmutations; an insertion of a single G residue within the RI 67 codonwill generate a novel frame shifted peptide (REPSQA) followed by a STOPcodon (TGA). The present inventors generated both a histidine missensemutation at Y98 (Y98H) and a frameshift mutation at R167 (R167fr) tocreate potentially immunogenic mutant VHL proteins that recapitulatefeatures of known VHL mutations. The frame shifted VHL protein willexpress a larger novel epitope and may thus be more immunogenic. Thesingle missense Y98H mutation will be a more stringent test of thisapproach since it entails a single amino acid change. These mutationswere introduced into the full-length mVHL sequence using both asite-specific mutagenesis protocol and PCR. Briefly, the R133 mutationwas created using specific PCR primers to introduce the mutation andpremature stop codon. This mutant, as well as wild-type (WT) VHL, wascloned into the yeast expression vector, pYEX-BX used for yeastexpression and into a mammalian expression vector pUP for transfectionand expression in melanoma cells. The Y64 point mutation was createdusing a site-specific mutagenesis protocol from Clontech that has shownprevious success.

The inserts were cloned into the yeast expression vector pYEX-BX andinto the mammalian expression vector pUP for transfection and expressionin melanoma cells. To achieve this goal, the inventors engineered yeastto express the VHL protein and tested the efficacy of the variousvaccine formulations in mice. The pYEX-BX plasmid contains acopper-inducible promoter that will permit controlled induction ofmurine VHL protein after transformation of S. cerevisiae.

The expression vectors harboring the VHL genes under control of theconstitutive CMV early promoter were transfected into B16 melanomacells. The cell lines grew in vitro and grew as tumors when injectedinto mice, confirming that the mutated VHL constructs were not bythemselves immunogenic or otherwise lethal to the transfected cells. Thefirst vaccination/tumor challenge experiment consisted of eighteen 6week old C57B6 mice being immunized by subcutaneous injection on day 0and day 7 with 20×10⁶ yeast expressing the R133 truncation mutant(VHLtrunc). On day 14, the mice were challenged with tumor bysubcutaneous injection as follows: 6 mice received 2.5×10⁴ untransfectedB16; 6 mice received 2.5×10⁴ B16 expressing VHLwt; 6 mice received2.5×10⁴ B16 expressing VHL VHLtrunc. The mice were evaluated for tumorgrowth 21 days post challenge. The results of this experiment areoutlined in Table 1 below.

TABLE 1 Tumor Growth Immunization Tumor Challenge (# mice with tumors)mVHLtrunc VAX B16 5/6 mVHLtrunc VAX B16 VHLwt 5/6 mVHLtrunc VAX B16VHLtrunc 0/6

These results showed that while the VHLtrunc vaccine (targeting a unique9 amino acids prior to truncation) provided protection from the B16 VHLtMut tumor challenge, the vaccine did not protect mice challenged withuntransfected B16 or B16 VHLwt. Therefore, the vaccination protocolinduces a powerful immune response, but this response may be limitedonly to the antigen against which the animals were vaccinated. However,because this truncated mutant generates a large sequence difference fromwild type VHL, it is possible that a more subtle mutation (i.e., onlyone residue) may produce an immune response to both mutant and wildtype.

In a second immunization/challenge experiment (Table 2), mice wereimmunized with either the wild-type VHL vaccine (mVHLwtVAX) or with thetruncated mutant VHL vaccine described above (mVHLtrunc VAX). The micewere divided into groups and challenged with untransfected B16, B16expressing wildtype VHL or B16 expressing the mutated VHL, as describedin the first experiment above. Results showed that again, immunizationwith the truncated VHL vaccine resulted in protection from tumorchallenge, and again confirmed that these mice were not protectedagainst challenge with wildtype tumor. Mice immunized with the wild-typetumor were not protected against challenge with the wildtype tumor,indicating that the vaccine did not break tolerance to the wildtypeprotein. However, when challenged with the mutated VHL-expressing tumor,50% of the mice immunized with wild-type protein were protected,indicating that the mutated VHL was recognized to some extent by themurine immune system. Given the specificity and efficacy of theyeast-based vaccine in these experiments, it will be a relatively simpletask to generate yeast targeting the most common mutations in humans,paving the way for a potential immunization approach as a therapeuticvaccine in humans.

TABLE 2 Tumor Growth Immunization Tumor Challenge (# mice with tumors)mock B16 3/3 B16 VHLwt 3/3 B16 VHLtrunc 2/3 mVHLwt VAX B16 5/6 B16 VHLwt5/6 B16 VHLtrunc 3/6 mVHLtrunc VAX B16 5/6 B16 VHLwt 4/5 B16 VHLtrunc0/6

Example 5

The following example demonstrates the use of a yeast-based vaccinecomprising a cancer antigen to treat breast cancer in vivo.

Most patients with early-stage cancers of solid organs, including lung,breast, and colon, can be cured by surgical removal of the primarytumor. Unfortunately, many patients present or relapse with hematogenousmetastases which, with rare exceptions, cannot be cured by currentlyavailable modalities, including surgery, radiation therapy,chemotherapy, or allogeneic stem cell transplantation (alloSCT).Likewise, although newer engineered cancer vaccines show significantpotency in animal models of recently established disease, once the tumorhas been established for more than 5 days or metastases have occurred,vaccines are generally ineffective as single agents (Borello et al.,2000, Blood 95:3011-3019) This is in part because tumor establishment istypically associated with induction of tolerance to tumor antigens,which must be broken to achieve successful therapy (Ye et al., 1994,Proc. Natl. Acad. Sci. USA. 91:3916-3920; Staveley-O'Carroll et al.,1998, Proc. Natl. Acad. Sci. USA 95:1178-1183). Vaccination aftermyeloablative alloSCT has produced incremental improvements but is stillunable to affect tumors established for more than 3 days (Anderson etal., 2000, Blood 95:2426-2433). Luznik et al., supra, incorporatedherein in its entirety, recently reported in a mouse breast cancer modelthat vaccination after a nonmyeloablative allogeneic stem celltransplantation (NST) protocol that achieves stable mixed bone marrowchimerism generates significantly enhanced tumor-specific immuneresponses capable of eliminating metastases 2 weeks after establishmentof the primary tumor without inducing graft-versus-host disease (GVHD).The significantly enhanced efficacy of this strategy relative tovaccination alone or vaccination after either autologous SCT or fullalloSCT depends on the action of both host and donor immune systems,which interact in the setting of mixed chimerism.

In the experiments of Luznik et al., the vaccine that was administeredconsisted of irradiated autologous tumor cells mixed withgranulocyte-macrophage colony stimulating factor (GM-CSF). In thefollowing experiment, the present inventors showed that a yeast-basedvaccine could substitute for the use of irradiated autologous tumorcells mixed with cells producing GM-CSF in the same animal model withequally efficacious results. In brief, the present inventors generated ayeast-based vaccine comprised of Saccharomyces cerevisiae yeasttransduced with a yeast expression vector encoding the gp70 protein ofthe mouse mammary tumor virus (MMTV) under the control of the CUP1promoter (Yeast gp70-IT). The gp70 protein is expressed in spontaneousbreast cancers that arise in Balb/c mice that are infected with MMTV.Following the protocol described by Luznik et al., Balb/c mice wereinjected subcutaneously with 10,000 4T1 tumor cells (Balb/c-derivedspontaneous breast cancer cells that express MMTV gp70) on day 0. Thesubcutaneous tumor was resected on day 13, prior to nonmyeloablativeallogeneic stem cell transplantation (NST) from MHC-compatible B10.D2donors. NST consisted of 200 cGy TBI on day 13, 10 million donor marrowcells intravenously on day 14, and cyclophosphamide 200 mg/kgintraperitoneally on day 17. Mice receiving B10.D2 marrow then receivedeither: (a) 20 million B10.D2 splenocytes on day 28 with no furthertreatment (No vaccine); (b) 20 million B10.D2 splenocytes on day 28 plusautologous tumor vaccine on day 31 (10⁶ irradiated 4T1 tumor cells mixedwith 5×10⁵ B78H1/GM-CSF, a GM-CSF-secreting, MHC-negative bystander cellline), or (c) 20 million B10.D2 splenocytes on day 28 plus theYeast-based gp70-IT vaccine of the present invention on day 31. As isreadily apparent in FIG. 5, the yeast-based vaccine of the presentinvention induced protection against fatal tumor recurrenceindistinguishable from protection induced by autologous tumor cellsproducing GM-CSF. The clinical usefulness of the yeast-based vaccineapproach of the present invention, as compared to using patientautologous tumor cells admixed with a bystander cell line producingGM-CSF, should be readily appreciated, and includes, but is not limitedto, the advantages of broader patient applicability, reduced variabilityof results, enhanced ability to design the vaccinating antigen, enhancedsafety, lack of necessity to include biological modifiers such as GM-CSFin the vaccine, etc.

Example 6

The following example demonstrates the use of a yeast-based vaccinecomprising a cancer antigen to treat a melanoma in vivo.

In this experiment, referring to Table 3, 5 groups of 5 mice each wereused. In Group A, mice received injections of PBS at 4 weeks and 2 weeksprior to tumor challenge, and 50×10⁶ yeast-based hMART-1 vaccine (yeastvehicle expressing human MART-1) at days 10 and 17 after tumorchallenge. In Group B, mice received injections of 50×10⁶ yeast-basedhMART-1 vaccine at 4 weeks and 2 weeks prior to tumor challenge and at10 and 17 days after tumor challenge. Group C mice received PBS at 4weeks and 2 weeks prior to tumor challenge and no administrations aftertumor challenge. Group D mice received injections of 50×10⁶ yeast-basedhMART-1 vaccine at 4 weeks and 2 weeks prior to tumor challenge and noadministrations after tumor challenge. Group E mice received 50×10⁶yeast-based EGFR vaccine (yeast vehicles expressing EGFR) at 4 weeks and2 weeks prior to tumor challenge and no administrations after tumorchallenge. At day 0, all mice received a tumor challenge of D16 melanomacells delivered subcutaneously. Mice in Groups A-D received 50,000 D16melanoma cells, which expressed endogenous mouse MART-1 (the cells werenot transfected with human MART-1), and the mice in Group E received50,000 D16 melanoma cells that had been transfected with EGFR.

TABLE 3 hMART-1 Vaccination −4 wk −2 wk 0 D10 D17 A (5) PBS PBS 50K B162-8 OD 2-8 OD B (5) 2OD 2OD 50K B16 2OD 2OD C (5) PBS PBS 50K B16 D (5)2OD 2OD 50K B16 E (5) 2OD EGFR 2OD EGFR 25-50K B16/ EGFR

The results are shown in FIG. 6. Mice in Groups B (immunized both beforeand after tumor challenge) and D (immunized before tumor challenge)showed significant reduction in tumor burden, demonstrating that theyeast vaccine expressing a melanoma antigen is effective againstmelanoma tumors, even across species.

Example 7

The following example demonstrates the construction of fusion proteinsfor expression in a yeast vehicle of the invention, wherein the fusionproteins comprise multiple immunogenic domains and multiple mutations ofthe same antigen.

The nucleotide and amino acid sequence for a variety of Ras familymembers are well known in the art. SEQ ID NO:2 is the nucleic acidsequence encoding human K-ras (also known in GenBank Accession No.NM_(—)033360). SEQ ID NO:2 encodes human K-ras, represented herein asSEQ ID NO:3. SEQ ID NO:4 is the nucleic acid sequence encoding murineK-ras (also known in GenBank Accession No. NM_(—)021284). SEQ ID NO:4encodes murine K-ras, represented herein as SEQ ID NO:5. SEQ ID NO:6 isthe nucleic acid sequence encoding human H-ras (also known in GenBankAccession No. NM_(—)005343). SEQ ID NO:6 encodes human H-ras,represented herein as SEQ ID NO:7. SEQ ID NO:8 is the nucleic acidsequence encoding murine H-ras (also known in GenBank Accession No.NM_(—)008284). SEQ ID NO:8 encodes murine H-ras, represented herein asSEQ ID NO:9. SEQ ID NO: 10 is the nucleic acid sequence encoding humanN-ras (also known in GenBank Accession No. NM_(—)002524). SEQ ID NO:10encodes human N-ras, represented herein as SEQ ID NO: 11. SEQ ID NO: 12is the nucleic acid sequence encoding murine N-ras (also known inGenBank Accession No. NM_(—)010937). SEQ ID NO:12 encodes human N-ras,represented herein as SEQ ID NO: 13.

FIG. 7 is a schematic drawing illustrating examples of fusion proteinscomprising multiple antigenic/immunogenic domains for use in ayeast-based vaccine of the present invention. In these exemplary fusionconstructs, amino acid positions 3-165 of a K-Ras protein (positions3-165 of SEQ ID NO:3) were used, which are also equivalent amino acidsin N-Ras and H-Ras (i.e., one could use positions 3-165 of N-Ras orH-Ras and achieve the same result). This sequence was then mutated atposition 12 to substitute a valine, cysteine or aspartic acid residuefor the glycine that normally occurs in this position (see GI-4014,GI-4015 and GI-4016, respectively), and at position 61 to substitute anarginine for the glutamine that normally occurs at this position. Asecond sequence was fused to (appended to) this sequence. The secondsequence is a domain from K-ras spanning amino acid positions 56-69 ofSEQ ID NO:3, which includes a mutation at position 61 to substitute aleucine for the glutamine residue that normally occurs at that position.Although these first three sequences are shown with the Q61L domainfused to the N-terminus of the longer sequence, other constructs havebeen produced in which the order of domains is reversed. The nucleotideand translated amino acid sequence for the construct encoding GI-4014are represented by SEQ ID Nos:14 and 15, respectively.

FIG. 7 also shows a multi-antigen Ras fusion vaccine (GI-4018), whichcontains all three of the position 12 mutations described above and bothof the position 61 mutations described above. The fusion protein wasconstructed as follows. A synthetic sequence comprising SEQ ID NO:1 isfollowed by four polypeptides which include various Ras mutations. Thefirst of the four depicted in FIG. 7 includes residues 3-30 of theN-terminus of K-Ras (SEQ ID NO:3), wherein the amino acid residue atposition 12 with respect to SEQ ID NO:3 has been mutated by substitutionof a valine for the glycine that naturally occurs at this position. Thesecond of the four domains includes amino acid residues 3-39 of SEQ IDNO:3), wherein the amino acid residue at position 12 with respect to SEQID NO:3 has been mutated by substitution of a cysteine for the glycinethat naturally occurs at this position. The third of the four domainsconsists of amino acid positions 3-165 of SEQ ID NO:3), which contains asubstitution of an aspartic acid for the glycine that normally occurs atposition 12 and a substitution of an arginine for the glutamine thatnormally occurs at position 61. The fourth of the four domains is adomain from K-ras spanning amino acid positions 56-69 of SEQ ID NO:3,which includes a mutation at position 61 to substitute a leucine for theglutamine residue that normally occurs at that position. Again, althoughthe domains are depicted in this order in FIG. 7, it is to be understoodthat the order of domains can be reorganized as desired.

This example is simply intended to be illustrative of how antigenconstructs useful in the present invention can be constructed. Similarstrategies using domains from different antigens, multiple domains fromthe same antigen, or repeated domains with different mutations, can beused for other antigens. This type of construct is particularly usefulwhen it is desirable to encompass several different mutations and/orcombinations of mutations that may occur at a single position in theantigen in nature, in a single vaccine construct.

All references cited herein are incorporated by reference in theirentireties.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1. A composition comprising: a) a yeast vehicle; and b) a recombinantfusion protein expressed by the yeast vehicle, the fusion proteincomprising the following amino acid sequences: i) an amino acid sequencecomprising positions 57-65 of a wild-type Ras protein having an aminoacid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11 or SEQ ID NO:13, except that, as compared to the wild-type Rasprotein, the glutamine at position 61 is mutated; and ii) an amino acidsequence comprising positions 3-165 of a wild-type Ras protein having anamino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11 or SEQ ID NO:13, except that, as compared to thewild-type Ras protein, the glycine at position 12 is mutated, and theglutamine at position 61 is mutated; wherein the amino acid sequence of(i) is appended to the N-terminus of the amino acid sequence of (ii). 2.The composition of claim 1, wherein the wild-type Ras protein comprisesthe amino acid sequence of SEQ ID NO:5.
 3. The composition of claim 1,wherein the wild-type Ras protein comprises the amino acid sequence ofSEQ ID NO:3.
 4. The composition of claim 1, wherein the fusion proteinfurther comprises a peptide linked to the N-terminus of the amino acidsequence of (i), the peptide consisting of at least two amino acidresidues that are heterologous to the amino acid sequence of (i),wherein the peptide stabilizes the expression of the fusion protein inthe yeast vehicle or prevents posttranslational modification of theexpressed fusion protein; wherein the amino acid residue at position oneof the fusion protein is a methionine; wherein the amino acid residue atposition two of the fusion protein is not a glycine or a proline;wherein none of the amino acid residues at positions 2-6 of the fusionprotein is a methionine; and, wherein none of the amino acid residues atpositions 2-5 of the fusion protein is a lysine or an arginine.
 5. Thecomposition of claim 4, wherein the peptide consists of between two andsix amino acid residues which are heterologous to the amino acidsequence of (i).
 6. The composition of claim 4, wherein the peptideconsists of two amino acid residues which are heterologous to the aminoacid sequence of (i).
 7. The composition of claim 1, wherein the fusionprotein further comprises a peptide linked to the N-terminus of theamino acid sequence of (i), the peptide consisting of at least two aminoacid residues that are heterologous to the amino acid sequence of (i),wherein the peptide stabilizes the expression of the fusion protein inthe yeast vehicle or prevents posttranslational modification of theexpressed fusion protein, and wherein the first six amino acids of thefusion protein consist of an amino acid sequence of M-X2-X3-X4-X5-X6;wherein M is methionine; wherein X2 is any amino acid except glycine,proline, lysine or arginine; wherein X3 is any amino acid exceptmethionine, lysine or arginine; wherein X4 is any amino acid exceptmethionine, lysine or arginine; wherein X5 is any amino acid exceptmethionine, lysine or arginine; and wherein X6 is any amino acid exceptmethionine.
 8. The composition of claim 7, wherein X6 is a proline. 9.The composition of claim 7, wherein the peptide consists of an aminoacid sequence of M-A-D-E-A-P (SEQ ID NO:1).
 10. The composition of claim1, wherein the yeast vehicle is selected from: a whole yeast, a yeastspheroplast, a yeast cytoplast, a yeast ghost, or a subcellular yeastmembrane extract or fraction thereof.
 11. The composition of claim 1,wherein the yeast vehicle is selected from: a whole yeast, a yeastspheroplast, a yeast cytoplast, or a yeast ghost.
 12. The composition ofclaim 1, wherein the yeast vehicle is a whole yeast or a yeastspheroplast.
 13. The composition of claim 1, wherein the yeast vehicleis a whole, killed yeast.
 14. The composition of claim 1, wherein theyeast vehicle is from a yeast selected from: Saccharomyces,Schizosaccharomyces, Kluveromyces, Hansenula, Candida or Pichia.
 15. Thecomposition of claim 1, wherein the yeast vehicle is from Saccharomycescerevisiae.
 16. The composition of claim 1, wherein the compositionfurther comprises a biological response modifier.
 17. The composition ofclaim 1, wherein the composition further comprises an immunopotentiator.18. The composition of claim 1, wherein fusion protein comprises: i) anamino acid sequence comprising positions 57-65 of the wild-type Rasprotein, except that, as compared to the wild-type Ras protein, theglutamine at position 61 is substituted with a leucine; and ii) an aminoacid sequence comprising positions 3-165 of the wild-type Ras protein,except that, as compared to the wild-type Ras protein, the glycine atposition 12 is substituted with a valine, and the glutamine at position61 is substituted with an arginine; wherein the amino acid sequence of(i) is appended to the N-terminus of the amino acid sequence of (ii).19. The composition of claim 18, wherein the wild-type Ras proteincomprises the amino acid sequence of SEQ ID NO:5.
 20. The composition ofclaim 18, wherein the wild-type Ras protein comprises the amino acidsequence of SEQ ID NO:3.
 21. The composition of claim 18, wherein thefusion protein further comprises a peptide linked to the N-terminus ofthe amino acid sequence of (i), the peptide consisting of at least twoamino acid residues that are heterologous to the amino acid sequence of(i), wherein the peptide stabilizes the expression of the fusion proteinin the yeast vehicle or prevents posttranslational modification of theexpressed fusion protein; wherein the amino acid residue at position oneof the fusion protein is a methionine; wherein the amino acid residue atposition two of the fusion protein is not a glycine or a proline;wherein none of the amino acid residues at positions 2-6 of the fusionprotein is a methionine; and, wherein none of the amino acid residues atpositions 2-5 of the fusion protein is a lysine or an arginine.
 22. Thecomposition of claim 21, wherein the peptide consists of between two andsix amino acid residues which are heterologous to the amino acidsequence of (i).
 23. The composition of claim 21, wherein the peptideconsists of two amino acid residues which are heterologous to the aminoacid sequence of (i).
 24. The composition of claim 18, wherein the yeastvehicle is selected from: a whole yeast, a yeast spheroplast, a yeastcytoplast, a yeast ghost, or a subcellular yeast membrane extract orfraction thereof.
 25. The composition of claim 18, wherein the yeastvehicle is selected from: a whole yeast, a yeast spheroplast, a yeastcytoplast, or a yeast ghost.
 26. The composition of claim 18, whereinthe yeast vehicle is a whole yeast or a yeast spheroplast.
 27. Thecomposition of claim 18, wherein the yeast vehicle is a whole, killedyeast.
 28. The composition of claim 18, wherein the yeast vehicle isfrom a yeast selected from: Saccharomyces, Schizosaccharomyces,Kluveromyces, Hansenula, Candida or Pichia.
 29. The composition of claim18, wherein the yeast vehicle is from Saccharomyces cerevisiae.
 30. Thecomposition of claim 18, wherein the composition further comprises abiological response modifier.
 31. The composition of claim 18, whereinthe composition further comprises an immunopotentiator.
 32. Thecomposition of claim 18, wherein the fusion protein comprises the aminoacid sequence of SEQ ID NO:15.
 33. The composition of claim 18, whereinthe fusion protein consists of the amino acid sequence of SEQ ID NO:15.34. The composition of claim 33, wherein the yeast vehicle is a whole,killed yeast.
 35. The composition of claim 33, wherein the yeast vehicleis from a yeast selected from: Saccharomyces, Schizosaccharomyces,Kluveromyces, Hansenula, Candida or Pichia.
 36. The composition of claim33, wherein the yeast vehicle is from Saccharomyces cerevisiae.
 37. Thecomposition of claim 34, wherein the yeast vehicle is from Saccharomycescerevisiae.
 38. The composition of claim 33, wherein the compositionfurther comprises a biological response modifier.
 39. The composition ofclaim 33, wherein the composition further comprises animmunopotentiator.
 40. The composition of claim 1, wherein fusionprotein comprises: i) an amino acid sequence comprising positions 57-65of the wild-type Ras protein, except that, as compared to the wild-typeRas protein, the glutamine at position 61 is substituted with a leucine;and ii) an amino acid sequence comprising positions 3-165 of thewild-type Ras protein, except that, as compared to the wild-type Rasprotein, the glycine at position 12 is substituted with a cysteine, andthe glutamine at position 61 is substituted with an arginine; whereinthe amino acid sequence of (i) is appended to the N-terminus of theamino acid sequence of (ii).
 41. The composition of claim 40, whereinthe wild-type Ras protein comprises the amino acid sequence of SEQ IDNO:5.
 42. The composition of claim 40, wherein the wild-type Ras proteincomprises the amino acid sequence of SEQ ID NO:3.
 43. The composition ofclaim 40, wherein the fusion protein further comprises a peptide linkedto the N-terminus of the amino acid sequence of (i), the peptideconsisting of at least two amino acid residues that are heterologous tothe amino acid sequence of (i), wherein the peptide stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein; whereinthe amino acid residue at position one of the fusion protein is amethionine; wherein the amino acid residue at position two of the fusionprotein is not a glycine or a proline; wherein none of the amino acidresidues at positions 2-6 of the fusion protein is a methionine; and,wherein none of the amino acid residues at positions 2-5 of the fusionprotein is a lysine or an arginine.
 44. The composition of claim 43,wherein the peptide consists of between two and six amino acid residueswhich are heterologous to the amino acid sequence of (i).
 45. Thecomposition of claim 43, wherein the peptide consists of two amino acidresidues which are heterologous to the amino acid sequence of (i). 46.The composition of claim 40, wherein the yeast vehicle is selected from:a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, ora subcellular yeast membrane extract or fraction thereof.
 47. Thecomposition of claim 40, wherein the yeast vehicle is selected from: awhole yeast, a yeast spheroplast, a yeast cytoplast, or a yeast ghost.48. The composition of claim 40, wherein the yeast vehicle is a wholeyeast or a yeast spheroplast.
 49. The composition of claim 40, whereinthe yeast vehicle is a whole, killed yeast.
 50. The composition of claim40, wherein the yeast vehicle is from a yeast selected from:Saccharomyces, Schizosaccharomyces, Kluveromyces, Hansenula, Candida orPichia.
 51. The composition of claim 40, wherein the yeast vehicle isfrom Saccharomyces cerevisiae.
 52. The composition of claim 40, whereinthe composition further comprises a biological response modifier. 53.The composition of claim 40, wherein the composition further comprisesan immunopotentiator.
 54. The composition of claim 1, wherein fusionprotein comprises: i) an amino acid sequence comprising positions 57-65of the wild-type Ras protein, except that, as compared to the wild-typeRas protein, the glutamine at position 61 is substituted with a leucine;and ii) an amino acid sequence comprising positions 3-165 of thewild-type Ras protein, except that, as compared to the wild-type Rasprotein, the glycine at position 12 is substituted with an asparticacid, and the glutamine at position 61 is substituted with an arginine;wherein the amino acid sequence of (i) is appended to the N-terminus ofthe amino acid sequence of (ii).
 55. The composition of claim 54,wherein the wild-type Ras protein comprises the amino acid sequence ofSEQ ID NO:5.
 56. The composition of claim 54, wherein the wild-type Rasprotein comprises the amino acid sequence of SEQ ID NO:3.
 57. Thecomposition of claim 54, wherein the fusion protein further comprises apeptide linked to the N-terminus of the amino acid sequence of (i), thepeptide consisting of at least two amino acid residues that areheterologous to the amino acid sequence of (i), wherein the peptidestabilizes the expression of the fusion protein in the yeast vehicle orprevents posttranslational modification of the expressed fusion protein;wherein the amino acid residue at position one of the fusion protein isa methionine; wherein the amino acid residue at position two of thefusion protein is not a glycine or a proline; wherein none of the aminoacid residues at positions 2-6 of the fusion protein is a methionine;and, wherein none of the amino acid residues at positions 2-5 of thefusion protein is a lysine or an arginine.
 58. The composition of claim57, wherein the peptide consists of between two and six amino acidresidues which are heterologous to the amino acid sequence of (i). 59.The composition of claim 57, wherein the peptide consists of two aminoacid residues which are heterologous to the amino acid sequence of (i).60. The composition of claim 54, wherein the yeast vehicle is selectedfrom: a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeastghost, or a subcellular yeast membrane extract or fraction thereof. 61.The composition of claim 54, wherein the yeast vehicle is selected from:a whole yeast, a yeast spheroplast, a yeast cytoplast, or a yeast ghost.62. The composition of claim 54, wherein the yeast vehicle is a wholeyeast or a yeast spheroplast.
 63. The composition of claim 54, whereinthe yeast vehicle is a whole, killed yeast.
 64. The composition of claim54, wherein the yeast vehicle is from a yeast selected from:Saccharomyces, Schizosaccharomyces, Kluveromyces, Hansenula, Candida orPichia.
 65. The composition of claim 54, wherein the yeast vehicle isfrom Saccharomyces cerevisiae.
 66. The composition of claim 54, whereinthe composition further comprises a biological response modifier. 67.The composition of claim 54, wherein the composition further comprisesan immunopotentiator.
 68. A composition comprising: a) a yeast vehicle;and b) a fusion protein expressed by the yeast vehicle, the fusionprotein comprising the amino acid sequence of SEQ ID NO:15, except thatas compared to SEQ ID NO:15, the glycine at position 25 is substitutedwith a cysteine.
 69. The composition of claim 68, wherein the yeastvehicle is selected from: a whole yeast, a yeast spheroplast, a yeastcytoplast, a yeast ghost, or a subcellular yeast membrane extract orfraction thereof.
 70. The composition of claim 68, wherein the yeastvehicle is selected from: a whole yeast, a yeast spheroplast, a yeastcytoplast, or a yeast ghost.
 71. The composition of claim 68, whereinthe yeast vehicle is a whole yeast or a yeast spheroplast.
 72. Thecomposition of claim 68, wherein the yeast vehicle is a whole, killedyeast.
 73. The composition of claim 68, wherein the yeast vehicle isfrom a yeast selected from: Saccharomyces, Schizosaccharomyces,Kluveromyces, Hansenula, Candida or Pichia.
 74. The composition of claim68, wherein the yeast vehicle is from Saccharomyces cerevisiae.
 75. Thecomposition of claim 68, wherein the composition further comprises abiological response modifier.
 76. The composition of claim 68, whereinthe composition further comprises an immunopotentiator.
 77. Acomposition comprising: a) a yeast vehicle; and b) a fusion proteinexpressed by the yeast vehicle, the fusion protein comprising the aminoacid sequence of SEQ ID NO:15, except that as compared to SEQ ID NO:15,the glycine at position 25 is substituted with an aspartic acid.
 78. Thecomposition of claim 77, wherein the yeast vehicle is selected from: awhole yeast, a yeast spheroplast, a yeast cytoplast, a yeast ghost, or asubcellular yeast membrane extract or fraction thereof.
 79. Thecomposition of claim 77, wherein the yeast vehicle is selected from: awhole yeast, a yeast spheroplast, a yeast cytoplast, or a yeast ghost.80. The composition of claim 77, wherein the yeast vehicle is a wholeyeast or a yeast spheroplast.
 81. The composition of claim 77, whereinthe yeast vehicle is a whole, killed yeast.
 82. The composition of claim77, wherein the yeast vehicle is from a yeast selected from:Saccharomyces, Schizosaccharomyces, Kluveromyces, Hansenula, Candida orPichia.
 83. The composition of claim 77, wherein the yeast vehicle isfrom Saccharomyces cerevisiae.
 84. The composition of claim 77, whereinthe composition further comprises a biological response modifier. 85.The composition of claim 77, wherein the composition further comprisesan immunopotentiator.
 86. The composition of claim 1, wherein fusionprotein comprises: i) an amino acid sequence comprising positions 57-65of the wild-type Ras protein, except that, as compared to the wild-typeRas protein, the glutamine at position 61 is substituted with a leucine;and ii) an amino acid sequence comprising positions 3-165 of thewild-type Ras protein, except that, as compared to the wild-type Rasprotein, the glycine at position 12 is substituted with an amino acidselected from the group consisting of a valine, a cysteine, and anaspartic acid, and the glutamine at position 61 is substituted with anarginine; wherein the amino acid sequence of (i) is appended to theN-terminus of the amino acid sequence of (ii).
 87. The composition ofclaim 86, wherein the wild-type Ras protein comprises the amino acidsequence of SEQ ID NO:5.
 88. The composition of claim 86, wherein thewild-type Ras protein comprises the amino acid sequence of SEQ ID NO:3.89. The composition of claim 86, wherein the yeast vehicle is selectedfrom: a whole yeast, a yeast spheroplast, a yeast cytoplast, a yeastghost, or a subcellular yeast membrane extract or fraction thereof. 90.The composition of claim 86, wherein the yeast vehicle is selected from:a whole yeast, a yeast spheroplast, a yeast cytoplast, or a yeast ghost.91. The composition of claim 86, wherein the yeast vehicle is a wholeyeast or a yeast spheroplast.
 92. The composition of claim 86, whereinthe yeast vehicle is a whole, killed yeast.
 93. The composition of claim86, wherein the yeast vehicle is from a yeast selected from:Saccharomyces, Schizosaccharomyces, Kluveromyces, Hansenula, Candida orPichia.
 94. The composition of claim 86, wherein the yeast vehicle isfrom Saccharomyces cerevisiae.
 95. The composition of claim 86, whereinthe composition further comprises a biological response modifier. 96.The composition of claim 86, wherein the composition further comprisesan immunopotentiator.